Conditioned ("use-dependent") inhibition by phenylalkylamines (PAAs) is a characteristic property of L- type calcium (Ca2+) channels. To determine the structural elements of the PAA binding domain we transferred sequence stretches of the pore-forming regions of repeat III and/or IV from the skeletal muscle alpha1 subunit (alpha1S) to the class A alpha1 subunit (alpha1A and expressed these chimeras together with beta1a and alpha2/delta subunits in Xenopus oocytes. The corresponding barium currents (IBa) were tested for PAA sensitivity during trains of depolarizing test pulses (conditioned block). IBa of oocytes expressing the alpha1A subunit were only weakly inhibited by PAAs (less than 10% conditioned block of IBa during a 100-ms pulse train of 0.1 Hz). Transfer of the transmembrane segment IVS6 from alpha1S to alpha1A produced an enhancement of PAA sensitivity of the resulting alpha1A/alpha1S chimera comparable to L-type alpha1 subunits (about 35% conditioned block Of IBa during a 100-ms pulse train of 0.1 Hz). Our results demonstrate that substitution of 11 amino acids within the segment RVS6 of alpha1A with the corresponding residues of alpha1S is sufficient to transfer L-type PAA sensitivity into the low sensitive class A Ca2+ channel.