The prolactin-producing rat anterior pituitary GH4C1 cells possess Ca2+-activated K channels which are activated by physiological elevations of the cytosolic Ca2+ concentration even at membrane potentials more negative than the normal level of about -50 mV. Whole-cell current recordings showed a marked outward tail current following depolarizing voltage steps to 0 mV from a holding potential close to the normal membrane potential. The half-time of this tail current was about 1.3 s after a 4-s depolarization step. The GH4C1 cells also possess voltage-activated Ca channels, and we conclude that this tail current is a Ca2+-activated K+ current for the following reasons: (1) The reversal potential for the tail current was close to the K+ equilibrium potential for a range of transmembrane K+ gradients. (2) The tail current was blocked by a Ca2+ antagonist, and the voltage dependence of this current closely mirrored the voltage dependence of the isolated Ca2+ current. The time-course of the decline of the tail current thus reflects the removal rate of the Ca2+ entering the cytosol through voltage-dependent Ca channels during the depolarizing voltage step. VIP stimulates prolactin secretion from GH4C1 cells, and this peptide prolonged the half-time of the tail current by about 47% in 63% of the cells. This indicates that VIP may prolong the transient cytosolic Ca2+ elevations following the action potentials in these cells. Such a mechanism might be an important factor for the control of the cytosolic Ca2+ level, and hence hormone secretion.