The electrophysiological properties of the mouse anterior pituitary cell line AtT-20/D16-16 were investigated with intracellular and patch-clamp techniques. Clonal AtT-20/D16-16 cells were found to be electrically excitable, with most cells exhibiting spontaneous bursting action potentials. The mean burst rates varied from 1.4 Hz at -55mV to 8.2 Hz at -25mV, showing an approximately linear frequency-current relationship in the low current range. The bursts consisted of one to several fast Na+ spikes superimposed on a slow pacemaker potential, followed by a Ca2+ spike and a Ca2+-sensitive afterhyperpolarization. Removal of either Na+ or Ca2+ from the bathing medium led to cessation of spontaneous activity and the appearance of arrhythmic firing patterns. Single channel recordings revealed the presence of Ca2+-dependent K+ channels with unitary conductances of approximately equal to 130 pS in physiological medium. These channels were activated by both intracellular Ca2+ and membrane depolarization. Addition of norepinephrine (10 microM) led to increases in burst frequency and beta-endorphin secretion mediated by activation of beta-adrenergic receptors. Our results, in conjunction with previous work, suggest that the Ca2+ that enters the cell during the burst may be involved in hormone secretion.