The role of cellular phosphatase activity in regulation of smooth muscle L-type Ca2+ channels was investigated using tautomycin, a potent and specific inhibitor of serin/threonin phosphatases type 1 and 2A. Tautomycin (1-100 nM) inhibited Ca2+ channel activity in smooth muscle cells isolated from human umbilical vein. Tautomycin-induced inhibition of Ca2+ channel activity was due to a reduction of channel availability which originated mainly from prolongation of the lifetime of unavailable states of the channel. Pretreatment of smooth muscle cells with the protein kinase inhibitor H-7 (10 microM) prevented the inhibitory effect of tautomycin. Our results suggest modulation of slow gating between available and unavailable states as a mechanism of phosphorylation-dependent down-regulation of Ca2+ channels in vascular smooth muscle.