The pore-forming alpha1 subunit of L-type voltage-gated Ca2+ channels is pharmacologically modulated by dihydropyridine (DHP) Ca2+ antagonists and agonists. Site-directed mutation of amino acids within transmembrane segments IIIS6 and IVS6 to those characteristic of DHP-insensitive channels revealed 2 mutations in IIIS6 (I1049F and I1052F) and 4 mutations in IVS6 (Y1365I, M1366F, I1372M, and I1373L) with increased KD values for (+)-[3H]PN200-110 binding. A tyrosine residue (Y1048) in IIIS6 that is conserved between DHP-sensitive and -insensitive Ca2+ channels was also altered by mutagenesis. Y1048F had a KD for (+)-[3H]PN200-110 binding that was increased 12-fold, and Y1048A had a KD at least 1000-fold higher than that of wild-type. These results support the hypothesis that transmembrane segments IIIS6 and IVS6 both contribute critical amino acid residues to the DHP receptor site and that Tyr-1048 within transmembrane segment IIIS6 is required for high affinity DHP binding, even though it is conserved between DHP-sensitive and -insensitive Ca2+ channels.