Long-term treatment of rat pituitary tumor cells with epidermal growth factor (EGF) inhibits 45Ca2+ uptake, intracellular calcium levels and subsequent prolactin secretion in response to membrane depolarization. In the present study we have used whole-cell voltage-clamp and single-channel patch-clamp recording to determine directly the effects of EGF (10 nM for 48 h) on L-type calcium current density, the current-voltage relationship, single-channel amplitude, and opening and closing dwell times in rat GH4C1 pituitary tumor cells. Sustained, nimodipine-sensitive inward currents (barium as the carrier) with an activation threshold of approximately -30 mV were elicited in both control and EGF-treated GH4C1 cells by depolarization. Mean current density normalized to membrane capacitance was reduced to 45% of control after EGF treatment. There was no difference in the voltage-dependent activation of L-type channels between control and EGF-treated cells. Analysis of single-channel current recordings showed that EGF treatment had no effect on unitary current amplitude or channel open and close durations. These results suggest that EGF reduces the number of voltage-gated calcium channels in GH4C1 cell membranes, which likely contributes to the decreased calcium uptake.