We examined the effects of the protein kinase C (PKC) inhibitor staurosporine (ST) on voltage-dependent K (KV) channels in rabbit coronary arterial smooth muscle cells. ST inhibited the KV current in a dose-dependent manner with a Kd value of 1.3 microM. The inhibition of the KV current by ST was voltage-dependent between -30 and +10 mV. The additive inhibition of the KV current by ST was voltage-dependent throughout the activation voltage range. The rate constants of association and dissociation of ST were 0.63 microM s and 0.92 s, respectively. ST produced use-dependent inhibition of the KV current. ST shifted the activation curve to more positive potentials but did not have any significant effect on the voltage dependence of the inactivation curve. ST did not have any significant effects on other types of K channel. Another PKC inhibitor, chelerythrine, and PKA inhibitor peptide (PKA-IP) had little effect on the KV current. These results suggest that ST interacts with KV channels that are in the closed state and that ST inhibits KV channels in the open state in a manner that is phosphorylation-independent and voltage-, time-, and use-dependent.