Prolactin (PRL) is involved in numerous biological processes in peripheral tissues and the brain. Though numerous studies have been conducted to elucidate the signal transduction pathways associated with the PRL receptor, very few have examined the role of ion conductances in PRL actions. We used the patch-clamp technique in "whole-cell" configuration and microspectrofluorimetry to investigate the effects of PRL on membrane ion conductances in the U87-MG human malignant astrocytoma cell line, which naturally expresses the PRL receptor. We found that a physiological concentration (4 nM) of PRL exerted a biphasic action on membrane conductances. Firstly, PRL activated a Ca²⁺-dependent K⁺ current which was sensitive to CTX and TEA. This current depended on PRL-induced calcium mobilization, through a JAK2-dependent pathway from a thapsigargin- and 2-APB-sensitive Ca²⁺ pool. Secondly, PRL also activated an inwardly-directed current, mainly due to the stimulation of calcium influx via nickel- and 2-APB-sensitive calcium channels. Both phases resulted in membrane hyperpolarizations, mainly through the activation of Ca²⁺-dependent K⁺ channels. As shown by combined experiments (electrophysiology and microspectrofluorimetry), the PRL-induced Ca²⁺ influx increased with cell-membrane hyperpolarization and conversely decreased with cell membrane depolarization. Thus, PRL-induced membrane hyperpolarizations facilitated Ca²⁺ influx through voltage-independent Ca²⁺ channels. Finally, PRL also activated a DIDS-sensitive Cl- current, which may participate in the PRL-induced hyperpolarization. These PRL-induced conductance activations are probably related to the PRL proliferative effect we have already described in U87-MG cells.