In voltage-dependent Ca2+ channels, alpha1 and beta subunits interact via two cytoplasmic regions defined as Alpha Interaction Domain (AID) and Beta Interaction Domain (BID). Several novel amino acids for that interaction have now been mapped in both domains by point mutations. It was found that three of the nine amino acids in AID and four of the eight BID amino acids tested were essential for the interaction. Whereas the important AID amino acids were clustered around five residues, the important BID residues were more widely distributed within a larger 16 amino acid sequence. The affinity of the AIDA GST fusion protein for the four interacting beta 1b BID mutants was not significantly altered compared with the wild-type beta 1b despite the close localization of mutated residues to disruptive BID amino acids. Expression of these interactive beta mutants with the full-length alpha 1A subunit only slightly modified the stimulation efficiency when compared with the wild-type beta 1b subunit. Our data suggest that non-disruptive BID sequence alterations do not dramatically affect the beta subunit-induced current stimulation.