The ACTH-secreting mouse AtT-20/D16–16 anterior pituitary tumour cell line was used to study adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) and protein kinase C (PKC) involvement in stimulus-secretion coupling pathways. In permeabilised AtT-20 cells under calcium ion-free conditions, forskolin (10 μm), CRH-41 (100 nm), guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S; 100 μm) but not mastoparan (10 μm) stimulated cAMP accumulation. Measurement of ACTH secretion under identical incubation conditions revealed that GTP-γ-S and mastoparan significantly stimulated ACTH secretion but forskolin and CRH-41 did not. This dissociates cAMP accumulation from ACTH secretion under calcium ion-free conditions and indicated that the effects of mastoparan and GTP-γ-S on ACTH secretion are not mediated by cAMP production. Calcium ions (1 nm to 1 mm) stimulated ACTH secretion from electrically permeabilised cells in a concentration-dependent manner. cAMP (100μm) and phorbol 12-myristate 13-acetate (PMA; 100 nm) synergistically enhanced the response to calcium ions. cAMP did not stimulate ACTH secretion in the absence of calcium ions nor did it alter the concentrations at which calcium stimulated ACTH secretion. This suggests that stimulation of ACTH secretion via the calcium-dependent pathway is necessary before any cAMP-mediated enhancement of secretion is manifest. PMA, however, did stimulate ACTH secretion in the absence of calcium ions, indicating distinct mechanisms for PKC-evoked secretion. Co-incubation with cAMP and PMA did not exceed the secretory response obtained with the combination of PMA and calcium ions. CRH-41 (1 pm to 100 nm) and forskolin (1 nm to 100μm) stimulated ACTH secretion from intact cells in a concentration-dependent manner. Co-incubation with PMA (100 nm) further enhanced the ACTH response to CRH-41 and forskolin; the effects were simply additive. The present study indicates that there are distinct roles for PKA and PKC in stimulussecretion coupling in AtT-20 cells. The PKA-dependent pathway, acting in concert with the calcium messenger system, serves as part of the stimulus-secretion coupling pathway by which activation of CRH-41 receptors control ACTH secretion. The PKC-dependent pathway, in contrast, seems to be independent of the calcium messenger system and may represent a separate control mechanism of ACTH secretion.