The cytoplasmic beta subunit of voltage-dependent calcium channels modulates channel properties in a subtype-specific manner and is important in channel targeting. A high affinity interaction site between the alpha1 interaction domain (AID) in the I-II cytoplasmic loop of alpha1 and the beta interaction domain (BID) of the beta subunit is highly conserved among subunit subtypes. We describe a new subtype-specific interaction (Ss1) between the amino-terminal cytoplasmic domain of alpha1A (BI-2) and the carboxyl terminus of beta4. Like the interaction identified previously () between the carboxyl termini of alpha1A and beta4 (Ss2), the affinity of this interaction is lower than AID-BID, suggesting that these are secondary interactions. Ss1 and Ss2 involve overlapping sites on beta4 and are competitive, but neither inhibits the interaction with AID. The interaction with the amino terminus of alpha1 is isoform-dependent, suggesting a role in the specificity of alpha1-beta pairing. Coexpression of beta4 in Xenopus oocytes produces a reduced hyperpolarizing shift in the I-V curve of the alpha1A channel compared with beta3 (not exhibiting this interaction). Replacing the amino terminus of alpha1A with that of alpha1C abolishes this difference. Our data contribute to our understanding of the molecular organization of calcium channels, providing a functional basis for variation in subunit composition of native P/Q-type channels.