Basic residues in the S4 segments of voltage-dependent channels and leucines within the heptad repeat motif in the S4-S5 region of Shaker potassium channels have been shown to have important influences on activation. Here we have compared the relative importance for activation of S4 arginines (mutated to neutral or negative residues) in each of the four repeats of a chimeric L-type calcium channel. Significant effects on midpoint potential and time constant of activation were produced by mutations in repeats I and III but not in repeats II and IV. Leucine or isoleucine mutations in repeats I and III had the same effect on the voltage dependence of calcium channel activation as the mutations at equivalent positions in the Shaker channel, indicating that the heptad motif plays a fundamental role in channel activation.