Membrane electrical properties of the clonal anterior pituitary cell (GH3) were studied using intracellular recording techniques. The resting potential in the GH3 cell was -48.0 .+-. 1.1 mV (mean .+-. SE, n = 39) in normal saline. The input resistance estimated from the linear portion of the current-voltage relationship was 453 .+-. 30 M.OMEGA. (n = 21). The maximum rate of rise of the action potential was 7.0 .+-. 1.1 V/s (n = 12) at room temperature (23-25.degree. C). The action potential had both Na and Ca components. The Ca component was abolished by addition of 4 mM Co2+. Sr2+ could substitute for Ca2+ in supporting spike initiation. As the concentration of Sr2+ was increased, the maximum rate of rise of the action potential increased. After replacement of Ca2+ with isomolar Ba2+ the membrane potential shifted to -6.1 .+-. 1.1 mV (n = 11). In the Ba solution, prolonged action potentials were evoked by a depolarizing current pulse after maintaining the membrane potential more negative than -50 mV. The release of both prolactin and growth hormone was enhanced by increasing the external K+ concentration to 50 mM in the presence of Ca2+. Sr2+ could substitute for Ca2+. Ba2+ enhanced the release of both hormones, even if the K+ concentration was unaltered. The facilitatory effects of high K+ and Ba2+ were markedly suppressed by addition of 2-4 mM Co2+. The potential-dependent increase in the membrane permeability to Ca2+, responsible for initiation of the Ca spike, plays a significant role in stimulation of hormone secretion in GH3 cells.