Treatment of rat anterior pituitary GH3 cells with caffeine causes a reversible enhancement of electrical activity superimposed over a depolarization of the plasma membrane potential. Similar results are obtained with theophylline, but not with isobutylmethylxanthine or forskolin. The effects of caffeine are not related to Ca2+ liberation from intracellular stores since they are not affected by incubation of the cells with ryanodine or thapsigargin. Furthermore, caffeine-induced hyperpolarization of the membrane is not detectable even in cells in which Ca2+ liberation from inositol 1,4,5-trisphosphate-sensitive compartments produces a prominent transient hyperpolarization in response to thyrotropin-releasing hormone. Reductions of Ca2+-dependent K+ currents caused by partial block of L-type Ca2+ channels by caffeine are not sufficient to explain the effects of the xanthine, since the results obtained with caffeine are not mimicked by direct blockade of Ca2+ channels with nisoldipine. GH3 cell inwardly rectifying K+ currents are inhibited by caffeine. Studies on the voltage dependence of the caffeine-induced effects indicate a close correlation between alterations of electrical parameters and reported values of steady-state voltage dependence of inactivation of these currents. We conclude that, as previously shown for thyrotropin-releasing hormone, modulation of inwardly rectifying K+ currents plays a major role determining the firing rate of GH3 cells and its enhancement by caffeine.