We studied the blocking mechanism of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the ATP-sensitive K+ channel in the single ventricular myocytes using the inside-out patch clamp technique. The channel activity in response to 5-hydroxydecanoate varied with each membrane patch corresponding to the sensitivity to ATP. In this condition the exogenous application of cAMP or cAMP-dependent protein kinase (PKA) obviously recovered the ATP-sensitive K+ channel activity after channel deactivation. By contrast, in membrane patches exhibited low sensitivity to ATP, endogenous cAMP-dependent protein kinase inhibitor (PKI) depressed the channel activity and restored the inhibitory action of 5-hydroxydecanoate and ATP on the channel. These results suggest that PKA-PKI system is involved in the regulatory mechanism of gating activity of the ATP-sensitive K+ channel and the blocking action of 5-hydroxydecanoate and ATP appears to be exerted by potentiating the inhibitory action of PKI on the channel.