The effects of protein kinase C (PKC) were studied on dihydropyridine (DHP)-sensitive Ca channels from rabbit skeletal muscle T tubule membranes. To determine which channel subunits become phosphorylated under the conditions used for electrophysiological studies, we first performed biochemical studies of phosphorylation. T tubular membranes were fused with vesicles of the lipid mixture used in the planar bilayers, and phosphorylation was assessed using the same concentrations of PKC, adenosine 5'-triphosphate, and buffers as were used in the electrophysiological experiments. The alpha 1 subunit of the DHP receptors was phosphorylated by PKC to an extent of 1 mol phosphate/mol protein. The beta subunit was also phosphorylated but to a significantly lesser extent. The DHP-sensitive Ca channel activity was studied after fusing T tubule membranes with planar bilayers (Ma, J., C. Mundiña-Weilenmann, M. M. Hosey, and E. Ríos. 1991. Biophys. J. 60:890–901). The bilayers were held at -80 mV and activated by depolarizing voltage clamp pulses. The observed Ca channels exhibited two open states (tau o1 = 5 ms and tau o2 = 25 ms). On addition of purified PKC to the intracellular side, the proportion of the longer open state increased threefold. The average open probability during a 2-s, maximally activating pulse (Pmax) increased from 10 to 15%. The voltage dependence of activation was not changed by PKC; the Boltzmann parameters were V1 = -20.5 mV and K = 10.5 mV, which were not significantly different from the reference channels. The deactivation (closing) time constant was increased from 7 to 12 ms after PKC.(ABSTRACT TRUNCATED AT 250 WORDS)