Prolactin (PRL) has been proposed to directly stimulate corticosterone release. However, the role of PRL on adrenocortical function in male HAA rats has not been fully clarified. The aim of this study was to investigate the effect of PRL on the secretion of corticosterone and progesterone using an in vitro cell culture system in male rats. Administration of PRL (10(-7) and 10(-6) M) resulted in dose-dependent increases in corticosterone and progesterone release. Cotreatment with PRL produced an increase in the stimulatory effects of ACTH-induced corticosterone and progesterone secretion. However, the PRL-induced corticosterone and progesterone releases were significantly reduced by treatment with AG490, a specific Janus kinase 2 (Jak2) inhibitor. In addition, administration of AG490 blunted the significant inhibition of ACTH-induced corticosterone and progesterone secretion by PRL. These results demonstrated that PRL could act directly on the adrenal gland to drive corticosterone and progesterone secretion in male rats. Additionally, the results emphasize that PRL stimulation of adrenal steroid release may be mediated through Jak2 activity.