The effects of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) on acute ACTH-stimulated cortisol secretion were assessed using a specific PKA inhibitor (H-89) and a PKC activator (phorbol 12-myristate 13-acetate, PMA) in dispersed head kidney cells of rainbow trout (Oncorhynchus mykiss). To investigate the sites of action of both PKA and PKC, pregnenolone (a cortisol precursor stemmed from the rate limiting step in cortisol synthesis) and 25-OH-cholesterol (an exogenous substrate that bypasses the rate limiting step) were used as substrates, with and without ACTH stimulation. Inhibition of PKA decreased ACTH-stimulated cortisol secretion while activation of PKC had the same effect, demonstrating that PKA stimulates and PKC inhibits cortisol synthesis. Inhibition of PKA and activation of PKC had no significant effect on pregnenolone-stimulated cortisol synthesis, indicating that both PKA and PKC act upstream from the pregnenolone step. Inhibition of PKA and activation of PKC had no significant effect on basal cortisol secretion in the presence of 25-OH-cholesterol, suggesting that PKA and PKC exert their effects on the mitochondrial cholesterol translocation step. This study provided evidence for the stimulatory role of PKA and the inhibitory role of PKC in the signalling pathways leading to cortisol synthesis in teleosts.