The response of the L-type Ca2+ current (ICa,L) in pituitary GH3 cells to variations in the action potential (AP) waveform was examined using the whole-cell configuration of the patch-clamp technique. ICa,L evoked during an AP waveform exhibited an early and a late component. The early component occurred on the rising phase of the AP; the late component coincided with the falling phase. Prolonging the falling phase of the AP increased the Ca2+ charge carried by ICa,L, although the amplitude of the late ICa,L was reduced. Prolonging the peak voltage of the AP waveform, however, increased the amplitude of the late component. ICa,L inactivated during a train of AP waveforms. When Ba2+ was used as the charge carrier, current inactivation during a train of APs decreased. Likewise, ICa,L evoked by the AP templates with irregular bursting pattern was inactivated. When the repetitive firing of APs with depolarizing potentials was replayed to cells, Ca2+ entry was not only spread over the entire AP, but also occurred during the interspike voltage trajectory. After application of thyrotropin releasing hormone (TRH; 10 microM), ICa,L in response to rectangular pulses was increased and the current/voltage relation shifted slightly to more negative values. TRH (10 microM), thapsigargin (10 microM) or cyclopiazonic acid (30 microM) enhanced the late component of the AP-evoked ICa,L. TRH also attenuated the inactivation of ICa,L during a train of APs. These results indicate that in pituitary GH3 cells, the time course and kinetics of ICa,L during the AP waveforms is distinct from that evoked by rectangular voltage clamp. Changes in the shape and firing pattern of APs in GH3 cells can modulate Ca2+ influx through L-type Ca2+ channels. Ca2+ release from internal stores may affect the magnitude of AP-evoked ICa,L in these cells.