The folates (vitamin B9) are a family of one-carbon donors required for biosynthetic processes in mammalian cells. Their absorption in the gut, transport to systemic tissues and across epithelia is mediated by three specific processes: (i) The proton-coupled folate transporter (PCFT-SLC46A1) mediates folate absorption across the apical brush-border membrane of the proximal small intestine and across the blood:choroid plexus:cerebrospinal fluid barrier. This process is electrogenic and optimal at pH ~5.5. As the pH increases the influx Kt increases and the Vmax decreases. (ii) The reduced folate carrier (RFC-SLC19A1) is ubiquitously expressed and is the major route of transport of folates into systemic tissues. RFC functions optimally at pH 7.4 and is an organic phosphate antiporter that produces large electrochemical-potential differences for folates across cell membranes. (iii) Folate receptors (FRα & FRβ) are GPI-anchored membrane glycoproteins that transport folates by an endocytic process. FRα is expressed at the apical (luminal) membrane of the renal proximal tubules, retinal pigment epithelial cells and the choroid plexus. FRβ is expressed in cells of hematopoietic origin. All the transporters are expressed in placenta; however, the absence of any one of these transporters, 00alone, does not impair placental function. There are gene-targeted mouse models for these transporters. RFC -/- is embryonic lethal; however, if the dams are folate-supplemented, the pups are normal at birth but die within two weeks due to atrophy of the hematopoietic organs. The FRβ -/- mouse has no phenotype. The FRα -/- mouse is embryonic lethal but when the dams are folate-supplemented pups are normal and their requirement for supplementation does not persist. Adult animals are fertile with a normal phenotype save for a slight folate deficiency. PCFT -/- mice are normal at birth but subsequently develop severe folate deficiency and neurological defects. The human counterparts include: (i) hereditary folate malabsorption, due to loss-of-function mutations in the PCFT gene, is characterized by signs and symptoms of severe systemic and neural folate deficiency, (ii) cerebral folate deficiency due to mutations in FRα resulting in neurological defects that, unlike in FRα -/- mice, manifest several years after birth. All three transporters are expressed in human cancers and have a role in cancer therapeutics. The classical antifolate, methotrexate – a dihydrofolate reductase inhibitor that causes tetrahydrofolate depletion, is transported via RFC with an influx Kt of ~ 5 µM. Resistance to this drug has been associated with loss-of-function mutations in, or loss of expression of, RFC. Recently, a second-generation much more potent antifolate of this class, pralatrexate, was approved for cancer treatment, with an influx Kt of 0.05 µM. Pemetrexed is another recently approved antifolate which, in its active (polyglutamate) forms synthesized in cells, is a direct inhibitor of one-carbon utilization in thymidylate and purine synthesis. While this agent has an affinity for RFC somewhat greater than MTX, its PCFT-mediated transport is better preserved than most other folates at neutral pH so that the drug’s antitumor activity is sustained even in the absence of RFC. A major emphasis of contemporary drug development in cancer therapeutics is on (i) identifying intracellular targets that drive proliferation of malignant cells and (ii) the development of “targeted” agents that selectively block these processes. Another approach involves the development of agents that utilize specific transporters that are primarily expressed and/or are functional in cancer, but not normal, cells. Since RFC is the major route of transport into normal tissues, it plays a major role in antifolate toxicity. On the other hand, while PCFT is ubiquitously expressed, it does not play an important role in the delivery of folates or antifolates into normal tissues at physiological pH. Drugs are being developed that have a very low affinity for RFC but a very high affinity for PCFT which operates more efficiency in the acid microenviroment of solid tumors. Folate receptors are highly expressed in a variety of epithelial tumors (FRα) and hematological malignancies (FRβ). In a different approach antineoplastics, structurally unrelated to folates, are coupled to folic acid through linkers that include a cleavable sulfhydryl bond. The complex is endocytosed and, when the reducing potential of the endosome is increased, the bond is broken and the drug, which must be lipid soluble, diffuses out of the endosome to reach its intracellular target. A similar strategy is being used to ablate macrophages, which express FRβ, for the treatment of inflammatory diseases.