The transient receptor potential channel TRPC3 is exclusively expressed in the apical membrane of principal cells of the collecting duct (CD) both in vivo and in the mouse CD cell line IMCD-3. Previous studies revealed that ATP-induced apical-to-basolateral transepithelial Ca(2+) flux across IMCD-3 monolayers is increased by overexpression of TRPC3 and attenuated by a dominant negative TRPC3 construct, suggesting that Ca(2+) entry across the apical membrane occurs via TRPC3 channels. To test this hypothesis, we selectively measured the Ca(2+) permeability of the apical membrane of fura-2-loaded IMCD-3 cells using the Mn(2+) quench technique. Mn(2+) influx across the apical membrane was increased 12- to 16-fold by apical ATP and was blocked by the pyrazole derivative BTP2, a known inhibitor of TRPC3 channels, with an IC(50) value <100 nM. In contrast, Mn(2+) influx was only increased approximately 2-fold by basolateral ATP. Mn(2+) influx was also activated by apical, but not basolateral, 1-stearoyl-2-acetyl-sn-glycerol (SAG), a known activator of TRPC3 channels. Apical ATP- and SAG-induced Mn(2+) influx was increased by overexpression of TRPC3 and completely blocked by expression of the dominant negative TRPC3 construct. Mn(2+) influx was also stimulated approximately 2-fold by thapsigargin applied to either the apical or basolateral side. Thapsigargin-induced flux was blocked by BTP2 but was unaffected by overexpression of TRPC3 or by dominant negative TRPC3. Apical ATP, but not basolateral ATP, increased transepithelial (45)Ca(2+) flux. These results demonstrate that the apical membrane of IMCD-3 cells has two distinct Ca(2+) influx pathways: 1) a store-operated channel activated by thapsigargin and basolateral ATP and 2) TRPC3 channels activated by apical ATP. Only activation of TRPC3 leads to net transepithelial apical-to-basolateral Ca(2+) flux. Furthermore, these results demonstrate that native TRPC3 is not a store-operated channel in IMCD-3 cells.