Micromolar concentrations of ATP stimulate biphasic change in transepithelial conductance across CaSki cultures, an acute increase (phase I response) followed by a slower decrease ( phase II response). Phase I and phase II responses involve two distinct calcium-dependent pathways, calcium mobilization and calcium influx. To test the hypothesis that phase I and phase II responses are mediated by distinct P2 purinergic receptors, changes in permeability were uncoupled by blocking calcium mobilization with 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA) or by lowering extracellular calcium, respectively. Under these conditions ATP EC50was 25 μM for phase Iresponse and 2 μM for phase II response. The respective agonist profiles were ATP > UTP > adenosine 5′- O-(3-thiotriphosphate) (ATP-γS) = N6-([6-aminohexyl]carbamoylmethyl)adenosine 5′-triphosphate (A8889) > GTP and UTP > ATP > GTP = A8889 > ATP-γS. Suramin blocked phase Iresponse and ATP-induced calcium mobilization, whereas pyridoxal phosphate-6-azophenyl-2′,4-disulfonic acid (PPADS) blocked phase II response and ATP-augmented calcium influx. ATP time course and pharmacological profiles for phase II response and augmented calcium influx were similar, with a time constant of 2 min and a saturable concentration-dependent effect (EC50of 2–3 μM). RT-PCR experiments revealed expression of mRNA for both the P2Y2and P2X4receptors. These results suggest that the ATP-induced phase I and phase IIresponses are mediated by distinct P2 purinergic receptor mechanisms.