The α1-subunits of the skeletal and cardiac L-type calcium channels (L-channels) contain nearly identical pore regions (P-regions) in each of the four internal homology repeats. In spite of this high conservation of the P- regions, native skeletal L-channels exhibit a unitary conductance that is only about half that of native cardiac L-channels. To identify structural determinants of this difference in L-channel conductance, we have characterized unitary activity in cell-attached patches of dysgenic myotubes expressing skeletal, cardiac, and chimeric L-channel α1-subunits. Our results demonstrate that the S5-S6 linker of repeat I (IS5-1S6 linker) is a critical determinant of the difference in skeletal and cardiac unitary conductance. The unitary conductances attributable to the wild-type skeletal (CAC6; ~14 pS) and cardiac (CARD1; ~25 pS) α1-subunits expressed in dysgenic myotubes are identical to those observed in native tissues. Chimeric α1-subunits containing skeletal sequence for the first internal repeat and all of the putative intracellular loops (SkC15), the IS5-IS6 linker and the intracellular loops (SkC51), or only the IS5-IS6 linker (SKC49) each exhibit a low, skeletal-like unitary conductance (≤17 pS). Constructs in which the IS5-IS6 linker is of cardiac origin (CARD1 and CSk9) display cardiac-like conductance (~25 pS). Unitary conductance and the rate of channel activation are apparently independent processes, since both SkC51 and SKC49 exhibit low, skeletal-like conductance and rapid, cardiac-like rates of ensemble activation. These results demonstrate that the IS5-IS6 linker strongly influences the single channel conductance of L-channels in a manner that is independent from the rate of channel activation.