Extracellular nucleotides regulate several components of the mucociliary clearance (MCC) process that removes noxious materials from the lung. Adenine and uridine nucleotides promote ion transport, coordinate cilia beating, and stimulate mucin secretion. While it is well established that extracellular ATP and other nucleotides promote MCC activities by activating cell surface purinergic receptors, it is largely unknown how nucleotides reach the extracellular space. Circumstantial evidence exists for a plasma membrane channel that conducts cytosolic nucleotides and for the involvement of a secretory pathway(s), but unambiguous proof of conductive or vesicular nucleotide release is lacking. Using radiolabeling and fluorescence HPLC-based approaches, adenosine, ATP/UTP, UDP, and UDP-glcuose, the naturally occurring agonists for the A2b, P2Y2, P2Y6, and P2Y14 receptors, respectively, were quantified in airway surface liquid with nanomolar sensitivity. Unlike short-lived ATP and UTP, UDP-glucose released into ASL was hydrolyzed only slowly. Nucleotides and UDP-sugars were released selectively to the mucosal surface of polarized Calu-3 cell monolayers. Since adenine nucleotides, UDP, and UDP-sugars participate in phosphorylation, sulfatation, and glycosylation reactions in the lumen of the Golgi apparatus, we hypothesize that apical surface expression of glycoproteins provides a mechanism for the vesicular release of nucleotides from airway epithelia. Electron-microscopy of Calu-3 cells revealed prominent sub-apical electro-lucent granules that resemble mucin-packed core-dense granules of goblet cells. In addition, confocal microscopy indicated MUC1 and MUC5AC immunoreactivity in the apical region of Calu-3 cells. Stimulation of mucin secretion with ionomycin resulted in enhanced release of nucleotides and UDP-sugars from Calu-3 cells as well as from A459, SPOC1 and HT-29 epithelial cells. Calu-3 cells labeled with FM 1-43, a water-soluble probe that becomes fluorescent upon binding to lipids, displayed robust ionomycin-promoted vesicle-plasma membrane recycling. These results suggest that nucleotides and nucleotide-sugars in the lumen of the secretory pathway are released to the extracellular environment via regulated exocytosis. In chronic lung diseases characterized by mucin-obstructed airways, UDP-sugar release from goblet cells may provide paracrine signaling to P2Y14 receptors on inflammatory cells