Interaction of defined regions within the intracellular, carboxyl terminal region of L-type calcium channels with CaM are believed to strongly influence the inactivation kinetics of the respective Ca²⁺ currents in vivo. Since the emphasis in previous experiments mainly lay on CaM interaction with the cardiac isoform a_1C, we aimed to investigate the highly homologous a1S (skeletal isoform) carboxyl terminus in terms of CaM binding. We expressed different regions of either the a_1C or the a_1S carboxyl terminus in E. coli and tested them for CaM binding using gel shift native PAGE in combination with mass spectrometry. No interaction between CaM and either isoform was observed in the absence of Ca²⁺ (2mM EDTA) arguing against a constitutive binding site for ApoCaM under our experimental conditions. Surprisingly, we were not able to detect a complex formation between CaM and the skeletal muscle isoform in the presence of calcium (within the range between 0.1–3 mM), while cardiac isoforms robustly interacted with CaM under these conditions. Data from the recently solved structure of the CaM complex of the cardiac carboxyl terminus (Van Petegem et al., 2005), allow the identification of residues within the interacting regions that are responsible for the diverse binding behaviour of the a_1C and a_1S carboxyl terminus.