The transmembrane segment IIIS5 of the L-type calcium channel alpha1 subunit participates in the formation of the 1,4-dihydropyridine (DHP) interaction domain (Grabner, M., Wang, Z., Hering, S., Striessnig, J., and Glossmann, H. (1996) Neuron 16, 207-218). We applied mutational analysis to identify amino acid residues within this segment that contribute to DHP sensitivity. DHP agonist and antagonist modulation of Ba2+ inward currents was assessed after coexpression of chimeric and mutant calcium channel alpha1 subunits with alpha2delta and beta1a subunits in Xenopus oocytes. Whereas DHP antagonists required Thr-1066, DHP agonist modulation crucially depended on the additional presence of Gln-1070 (numbering according to alpha1C-a), which also further increased the sensitivity to DHP antagonists. Asp-955, which is found at the corresponding position in the calcium channel alpha1S subunit from carp skeletal muscle, displayed functional similarity to Gln-1070 with respect to DHP interaction. We conclude that these residues (Thr-1066 plus Gln-1070 or Asp-955), which are located in close vicinity on the same side of the putative alpha-helix of transmembrane segment IIIS5, form a crucial DHP binding motif.