Pituitary lactotrophs fire action potentials spontaneously and the associated voltage-gated calcium influx is sufficient to maintain high prolactin release. Here we studied the role of hyperpolarization-activated cation channels in pacemaking activity, calcium signaling, and prolactin secretion in these cells. A slowly developing and hyperpolarization-activated inward current was identified but only in a fraction of lactotrophs. The current was blocked by ZD7288, a relatively specific blocker of these channels. However, the pacemaking activity increased in ZD7288-treated cells independently of the presence of this current. This in turn facilitated voltage-gated calcium influx and transiently stimulated prolactin secretion. Sustained ZD7288 application in concentrations that are commonly used to block the hyperpolarization-activated cation channels inhibited hormone release at elevated intracellular calcium concentrations. Agonist and Bay K 8644-stimulated prolactin release was also inhibited by ZD7288, indicating that this compound attenuates the exocytotic pathway downstream of calcium influx. (c) 2006 Elsevier Inc. All rights reserved.