Contribution of prostaglandins D2, E2 and I2 (PGD2, PGE2 and PGI2) on the regulation of ATP-sensitive K⁢ channel (KATP channel) was investigated in isolated single rat ventricular cardiac myocytes using the patch clamp technique. PGD2, PGE2 and PGI2 did not affect KATP channel activity in the inside-out patch, but increased channel activity in a dose-dependent manner when the channel activities were attenuated by the administration of 100 μM ATP to the internal solution in the inside-out patch. Channel activations by the prostaglandins were abolished by 50 μM glibenclamide, a KATP channel blocker. Dose-response curves of relative channel activity against the ATP concentrations of internal solution in the inside-out patch were shifted to the right in the presence of those three prostaglandins. The rank order of the channel stimulatory potencies (as IC50 for ATP) calculated from the dose-response curves were PGI2 ＞ PGD2 ＞ PGE2. Conductance of the channel was not changed by those three prostaglandins. In conclusion, we suggest that prostaglandins D2, E2 and I2 are involved in the regulation of KATP channel activity in certain circumstances, and that those three prostaglandins may cause myocardial relaxation by opening KATP channels, thus protecting the heart from ischema.