1. The inactivation of high-voltage-activated (HVA) calcium currents during long depolarizations in holding potential (Vh) was studied with the use of whole cell patch-clamp recording from rat anterior pituitary cells. 2. An ultra-slow inactivation in the amplitude of HVA calcium currents, with an average slow time constant of 149.3 s for peak currents and 159.1 s for sustained currents (n = 9), was unveiled during 5-min step depolarizations in Vh. 3. The ultra-slow inactivation of HVA calcium currents was found to be generated by at least two processes: a voltage-dependent inactivation that increases with increasing depolarization in Vh and an activity-dependent inactivation that is initiated, but not increased, with increasing depolarization in Vh. The relative contribution of the activity-dependent component to the ultra-slow inactivation was 80% when Vh was stepped from -80 to -60 mV and only 40% when Vh was stepped from -80 to -40 mV. 4. The activity-dependent inactivation of the HVA currents was not altered significantly in experiments in which barium replaced calcium as charge carrier and 1,2-bis (1-aminophenoxy) ethane N,N,N' N'-tetraacetic acid (BAPTA) was used as an intracellular calcium buffer instead of the less potent ethylenglycol-bis-(beta-aminoethylether) N,N,N' N'-tetraacetic acid (EGTA). In addition, activity-dependent inactivation was observed with sodium as the charge carrier through the calcium channels. 5. The activity-dependent inactivation depends on divalent cation influx. The activity-dependent inactivation was abolished when the test potentials, during the depolarization in Vh, were increased from 0 to +70 mV (close to the reversal potential for calcium currents under our experimental conditions). This reduction in driving force for calcium currents eliminated divalent cation influx and abolished the activity-dependent inactivation. 6. The activity-dependent inactivation lacks several characteristic features for calcium-dependent inactivation, such as dependence on charge carrier (see above), dependence on the size of the calcium current, and increase in decay rate of the calcium current during the test pulse. These latter notions were also supported by our paired pulse experiments, in which the calcium current elicited by a constant test pulse was virtually unaffected (7%) by conditioning pulses that produced maximal calcium currents. We therefore conclude that the dependence of activity-dependent inactivation on divalent cation influx cannot be attributed to the known form of calcium-dependent inactivation. 7. In conclusion, this study shows that calcium influx through HVA channels in anterior pituitary cells can be regulated by subthreshold changes in membrane potential and that the extent of this regulation depends on low-frequency activation of HVA calcium channels during the depolarization in membrane potential. Thus the pituitary cell may regulate hormone secretion by changes in membrane potential and in a use-dependent manner via regulation of calcium influx.