The effects of a long-term blockade of L-type Ca2+ channels on membrane currents and on the number of dihydropyridine binding sites were investigated in skeletal muscle fibers. Ca2+ currents (ICa) and intramembrane charge movement were monitored using a voltage-clamp technique. The peak amplitude of ICa increased by more than 40% in fibers that were previously incubated for 24 hr in solutions containing the organic Ca2+ channel blocker nifedipine or in Ca2+-free conditions. A similar incubation period with Cd2+, an inorganic blocker, produced a moderate increase of 20% in peak ICa. The maximum mobilized charge (Qmax) increased by 50% in fibers preincubated in Ca2+-free solutions or in the presence of Cd2+. Microsomal preparations from frog skeletal muscle were isolated by differential centrifugation. Preincubation with Cd2+ prior to the isolation of the microsomal fraction doubled the number of 3H-PN200-110 binding sites and produced a similar increase in the values of the dissociation constant. The increase in the number of binding sites is consistent with the increase in the peak amplitude of ICa as well as with the increase in Qmax.