The transient removal of dopamine (DA) selectively potentiated the prolactin (PRL) releasing action of thyrotropin-releasing hormone (TRH) but not vasoactive intestinal peptide (VIP). Consistent with these findings, the PRL-stimulating actions of agents which activated the Ca2+/protein kinase C second messenger pathway but not the adenylate cyclase system were also potentiated. In the current study we have extended these findings to determine the second messenger system mediating the potentiating action of the removal of DA. Dispersed anterior pituitary cells from E2-treated Sprague-Dawley rats were cultured on plastic coverslips. Cells tonically superfused with DA (500 nM were challenged with TRH (100 nM) 20 min after no additional treatment or a 10-min treatment with 8-Br-cyclic adenosine monophosphate (8-Br-cAMP), the Ca2+ ionophore A23187,12–0-tetradecanoyl-phorbol-13-acetate (TPA), TRH, or VIP. The potentiation of the TRH response was compared to the 4- to 5-fold potentiation observed following the removal of DA for 10 min. 8-Br-cAMP at the concentration used (500 µM) was unable to alter the basal rate of PRL release, but, as VIP (500 nM), potentiated 2- to 3-fold the PRL-releasing action of TRH. A prior administration of TRH (100 nM) did not affect the responsiveness of the cells to a second challenge with TRH 20 min later. Both A23187 (20 µM) and TPA (5 or 50 nM) induced a sustained rise in the rate of PRL release. TPA-treated cells showed an increased responsiveness to TRH, whereas A23187-treated cells did not. These results suggest that increasing cAMP levels either by VIP or 8-Br-cAMP in part mimics the potentiation caused by the transient removal of DA. Although TPA potentiated the PRL-releasing action of TRH, stimulating the influx of Ca2 + with the ionophore A23187 or the presumed activation of Ca2+/protein kinase C pathway by a priming treatment with TRH did not. Therefore, the mechanism by which TPA is potentiating the response to TRH is unclear. The finding that prior removal of DA or exposure to VIP can potentiate the action of TRH supports the concept that a secretory response can represent the integration of the action of multiple regulatory neurohormones delivered in a specific sequence, a so-called ‘pleotropic regula