Voltage-activated calcium channels in the plasma membrane of excitable cells play a major role in regulating the intracellular concentration of calcium. In this way they transduce the electrical effects of modulating other ion channels into a chemical signal that can alter cell function. Recently, the predominant calcium channel in a wide variety of vertebrate cell types has been characterized with patch-clamp techniques. Like many other ion channels, these dihydropyridine- sensitive calcium channel are modulated by cAMP -dependent protein phosphorylation. We have investigated the phosphorylation dependence of individual dihydropyridine-sensitive channels under voltage-clamp in cell-free patches of native membrane from a rat pituitary tumor cell line (GH3). Our data suggest that the enzymatic addition or removal of phosphate esters on the channel protein by endogenous kinases and phosphatases profoundly alters the response of these channels to depolarization of the membrane. Experiments with exogenous protein kinases and their inhibitors, purified to homogeneity by affinity chromatography, support that conclusion. Phosphorylation by the cAMP-dependent kinase leads to short bursts of openings with an average duration of -1 ms. Subsequent phosphorylation by the calcium/calmodulin-dependent protein kinase type II produces much longer openings of -10 ms duration, like those produced by BAY k 8644. In the absence of ATP-Mg., the kinases have no effect and the channel opens very rarely, if at all, in patches of native membrane. Additional data suggest that the phosphorylation dependence of calcium channel activity underlies both the modulation by dihydropyrindines and the rapid inactivation produced by intracellular accumulation of calcium, to additional properties that distinguish these channels from other voltage-activated calcium channels.