Hormonal stimulation of voltage-dependent Ca2+ channels in pituitary cells is thought to contribute to the sustained phase of Ca2+ entry and secretion induced by secretion stimulating hormones and has been suggested as a mechanism for refilling the Ca2+ stores. Using the cell-attached patch-clamp technique, we studied the stimulation of single Ca2+ channels by thyrotropin-releasing hormone (TRH) in rat GH3 cells. We show that TRH applied from the bath switched the activity of single L-type Ca2+ channels from a gating mode with very low open probability (po) to a gating mode with slightly smaller conductance but 10 times higher po. Interconversions between these two gating modes were also observed under basal conditions, where the equilibrium was shifted towards the low po mode. TRH applied from the pipette had no effect, indicating the involvement of a cytosolic compound in the stimulatory pathway. We show that TRH does not potentiate all the L-type Ca2+ channels in a given membrane patch and report evidence for co-expression of two functionally different L-type Ca2+ channels. Our results uncover the biophysical mechanism of hormonal stimulation of voltage-dependent Ca2+ channels in GH3 cells and are consistent with differential modulation of different subtypes of dihydropyridine-sensitive Ca2+ channels.