The classical secretagogues, adrenocorticotrophic hormone (ACTH) and angiotensin II (AII), are known to stimulate cortisol production from bovine adrenocortical zona fasciculata/reticulariscells. This is seen in both freshly isolated, enzymically dispersed, cells and in primary cultures prepared from these cells. Previous published work has also shown that adrenergic agonists stimulate steroidogenesis in primary cultures of bovine adrenocortical cells, but not in freshly isolated cells. In addition, cholinergic agonists have been shown to stimulate steroidogenesis in both freshly isolated cells, and in cells in primary culture, and that this process is linked to increased turnover of inositol containing phospholipids. Initial characterisation of primary cultures of bovine adrenocortical zona fasciculata/reticularis cells, prepared by a collagenase digestion procedure, showed that both ACTH and AII stimulated cortisol production from both freshly isolated and cultured cells. Adrenergic agonists stimulated cortisol production in cultured cells, but failed to stimulate cortisol production from freshly isolated cells. Cholinergic agonists stimulated cortisol production from both freshly isolated and cultured cells. Using specific alpha and beta adrenergic agonists and antagonists it was shown that adrenergic stimulation of cortisol production from cultured cells was mediated by beta-adrenergic receptors. Schild analysis, using specific beta-adrenergic antagonists, showed that beta₁- adrenoceptors were responsible for this. Adrenergic agonists were also shown to increase intracellular cyclic AMP levels in cultured cells which was directly responsible for stimulating steroidogenesis. These agonists had no effect on turnover of cellular phosphoinositides. Using specific cholinergic agonists and antagonists, it was shown that M₃-cholinergic receptors were responsible for mediating stimulation of cortisol production from cultured cells produced by cholinergic agonists. It was also shown that cholinergic agonists activated a phospholipase-C-dependent breakdown of phosphoinositides, and that this was responsible for initiating steroidogenesis. Cholinergic agonists had no effect on cellular cyclic AMP levels.