The autosomal dominant polycystic kidney disease (ADPKD) genes polycystin-1 (PC1) and polycystin-2 (PC2) colocalize with other polycystic kidney disease genes in the apical monocilium of mouse and dog renal epithelial cells. PC1-deficient, precystic, embryonic mouse collecting duct cells in primary culture show impaired PC2-dependent ciliary mechanosensation. However, most human ADPKD kidneys express normal or elevated levels of apparently full-length PC1 and PC2 polypeptides. We have localized PC1 and PC2 in the monocilium of normal human renal (NK) epithelial cells in confluent primary culture. However, cilia of ADPKD cyst cells from a patient with the novel PC1 mutation ΔL2433 were devoid of detectable PC1, and 70% of cilia lacked PC2, despite normal total cell levels of both polypeptides. 34% of confluent NK cell coverslips exposed to ‘physiological’ laminar shear stress of 0.75 or 2.3 dyn cm^-2, respectively, increased [Ca²⁺]_i by 56+10 nM (n=22) or 48+9 nM (n=13) above resting levels (149+6 nM, n=61; P<0.01 for both). 80% of confluent NK coverslips exposed to 'diuretic' or supraphysiologic shear stresses of 10 or 35 dyn cm^-2, respectively, increased [Ca²⁺]_i by 207+65 nM (n=13) or 252+43 nM (n=15, P< 0.0001 for both). This flow-induced, transient [Ca²⁺]_i increase required both extracellular Ca²⁺ and release from intracellular Ca²⁺ stores, and was inhibited by 3 μM GsMTx-IV, 30 μM ryanodine and 20 μM 2-APB, but not by phospholipase C inhibitor U73122 (10 μM). ADPKD cyst cells, in contrast, lacked flow-sensitive [Ca²⁺]_i signalling and exhibited reduced ER Ca²⁺ stores and store-depletion-operated Ca²⁺ entry, but retained near-normal Ca²⁺_i responses to angiotensin II and to vasopressin. Expression of wildtype and mutant CD16.7-PKD1(115-226) fusion proteins revealed within the C-terminal 112 aa of PC1 a ciliary targeting signal active in NK and cyst cells, and independent of the coiled-coil domain. However, the coiled-coil domain was required for CD16.7-PKD1(115-226) expression to accelerate decay of the flow-induced Ca²⁺ signal in NK cells. These data provide evidence for ciliary dysfunction in human ADPKD, and support a role for that dysfunction in disease pathogenesis.