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 EC₅₀ was 25 µM for phase I response and 2 µM for phase II response. The respective agonist profiles were ATP > UTP > adenosine 5'-O-(3-thiotriphosphate) (ATP-S) = N⁶-([6-aminohexyl]carbamoylmethyl)adenosine 5'-triphosphate (A8889) > GTP and UTP > ATP > GTP = A8889 > ATP-S. Suramin blocked phase I response 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 (EC₅₀ of 2-3 µM). RT-PCR experiments revealed expression of mRNA for both the P2Y₂ and P2X₄ receptors. These results suggest that the ATP-induced phase I and phase II responses are mediated by distinct P2 purinergic receptor mechanisms.