The electrical and pharmacological properties of protein kinase C (PKC) and its effect on the single Ca2+-activated K+ channel (Kca-channel) in the cultured smooth muscle cells of rat mesenteric artery were studied using a patch-clamp technique. The Kca-channel had a slope conductance of 151+/-7 pS (mean+/-S.E.) in symmetrical 142 mm K solutions. The high conductance K+ channel, applied to the outer side of membrane patches, was potently inhibited by charybdotoxin (0.1 microM) and tetraethylammonium (0.5 microM), but not by apamin (0.4 microM). In cell-attached patches, bath application of phorbol 12-myristate 13-acetate (PMA, 2 microM), a PKC activator, inhibited the activity of the Kca-channel in the presence of the Ca2+ ionophore, A 23187 (10 microM). This inhibition was reversed by subsequent application of staurosporine (1 nM), a PKC inhibitor. Application of 1-oleoyl-2-acetylglycerol (OAG, 30 microM), another PKC activator, also inhibited the A 23187-induced activation of the K+ channel, and this inhibition was reversed by staurosporine. In inside-out patches, bath application of PKC (0.2 munits), in the presence of ATP (1 mM) and PMA (1 microM), inhibited the K+ channel. These results indicate that protein kinase C inhibits the Ca2+-activated K+ channel of mesenteric artery smooth muscle cells in the rat.