The role of protein kinase C (PKC) in cellular regulation of L-type Ca2+ channels was investigated in human umbilical vein smooth muscle. Activation of PKC, by low concentrations (< 30 nM) of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) caused inhibition of Ca2+ channels, while higher concentrations of TPA (> 100 nM) elicited a transient rise, followed by sustained inhibition of Ca2+ channel activity in cell-attached patches. Low TPA concentrations predominantly reduced channel availability, while high concentrations of TPA (100 nM) transiently increased channel availability and, in addition, prolonged mean open time. The inactive 4-alpha-phorbol-12,13- didecanoate failed to affect channel activity, and pretreatment of the cells with PKC inhibitors (H-7, chelerythrine) antagonized inhibitory and stimulatory effects of TPA. Our results provide evidence for two distinct PKC-dependent mechanisms of L-type Ca2+ channel regulation in smooth muscle.