The influences of specific protein phosphatase and protein kinase inhibitors on the ATP-sensitive K⁢ (KATP) channel-opening effect of pinacidil were investigated in single rat ventricular myocytes using patch clamp technique. In cell-attached patches, pinacidil (100 μM) induced the opening of the KATP channel, which was blocked by the pretreatment with H-7 (100 μM) whereas enhanced by the pretreatment with genistein (30 μM) or tyrphostin A23 (10 μM). In inside-out patches, pinacidil (10 μM) activated the KATP channels in the presence of ATP (0.3 mM) or AMP-PNP (0.3 mM) and in a partial rundown state. The effect of pinacidil (10 μM) was not affected by the pretreatment with protein tyrosine phosphatase 1B (PTP1B, 10 μg ml⁣1), but blocked by the pretreatment of protein phosphatase 2A (PP2A, 1 U ml⁣1). In addition, pinacidil (10 μM) could not induce the opening of the reactivated KATP channels in the presence of H-7 (100 μM) but enhanced it in the presence of ATP (1 mM) and genistein (30 μM). These results indicate that the KATP channel-opening effect of pinacidil is not mediated via phosphorylation of KATP channel protein or associated protein, although it still requires the phosphorylation of serine/threonine residues as a prerequisite condition.