In cardiac myocytes, LTCCs (L-type calcium channels) form a functional signalling complex with ryanodine receptors at the JM (junctional membrane). Although the specific localization of LTCCs to the JM is critical for excitation-contraction coupling, their targeting mechanism is unclear. Transient transfection of GFP (green fluorescent protein)-α(1S) or GFP-α(1C), but not P/Q-type calcium channel α(1A), in dysgenic (α(1S)-null) GLT myotubes results in correct targeting of these LTCCs to the JMs and restoration of action-potential-induced Ca2+ transients. To identify the sequences of α(1C) responsible for JM targeting, we generated a range of α(1C)-α(1A) chimaeras, deletion mutants and alanine substitution mutants and studied their targeting properties in GLT myotubes. The results revealed that amino acids L(1681)QAGLRTL(1688) and P(1693)EIRRAIS(1700), predicted to form two adjacent α-helices in the proximal C-terminus, are necessary for the JM targeting of α(1C). The efficiency of restoration of action-potential-induced Ca2+ transients in GLT myotubes was significantly decreased by mutations in the targeting motif. JM targeting was not disrupted by the distal C-terminus of α(1C) which binds to the second α-helix. Therefore we have identified a new structural motif in the C-terminus of α(1C) that mediates the targeting of cardiac LTCCs to JMs independently of the interaction between proximal and distal C-termini of α(1C).