Expression of cardiac L-type Ca2+ channels in dysgenic myotubes results in large Ca2+ currents and electrically evoked contractions resulting from Ca2+-entry dependent release of Ca2+ from the sarcoplasmic reticulum. By contrast, expression of either P/Q-type or N-type Ca2+ channels in dysgenic myotubes does not result in electrically evoked contractions despite producing comparably large Ca2+ currents. In this work we examined the possibility that this discrepancy is caused by the preferential distribution of expressed L-type Ca2+ channels in close apposition to sarcoplasmic reticulum Ca2+ release channels. We tagged the N termini of different alpha1 subunits (classes A, B, C, and S) with a modified green fluorescent protein (GFP) and expressed each of the fusion channels in dysgenic myotubes. Each GFP-tagged alpha1 subunit exhibited Ca2+ channel activity that was indistinguishable from its wild-type counterpart. In addition, expression of GFP-alpha1S and GFP-alpha1C in dysgenic myotubes restored skeletal- and cardiac-type excitation-contraction (EC) coupling, respectively, whereas expression of GFP-alpha1A and GFP-alpha1B failed to restore EC coupling of any type. Laser-scanning confocal microscopy revealed a distinct expression pattern for L-type compared with non-L-type channels. After injection of cDNA into a single nucleus, GFP-alpha1S and GFP-alpha1C were present in the plasmalemma as small punctate foci along much of the longitudinal extent of the myotube. In contrast, GFP-alpha1A and GFP-alpha1B were not concentrated into punctate foci and primarily were found adjacent to the injected nucleus. Thus, L-type channels possess a targeting signal that directs their longitudinal transport and insertion into punctate regions of myotubes that presumably represent functional sites of EC coupling.