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Cells isolated from the zona fasciculata/reticularis (ZFR) of the bovine adrenal cortex and maintained in culture were found to secrete cortisol in response to vasopressin stimulation. The increased cortisol secretion was dose dependent, with a threshold response at 1 nM and a maximal response (1·68-fold over basal) at 0·1 µM. In cells cultured in the presence of [³H]inositol (to prelabel the membrane phosphoinositide pool), stimulation with vasopressin in the presence of LiCl (10 mM) resulted in a similar dose-dependent increase in labelling of the phosphoinositol fraction, with a maximal response (1·45-fold over basal) at 10 nM. The increased labelling of the phosphoinositol fraction was independent of extracellular Ca²⁺ as it was not abolished in medium with [Ca²⁺ ] buffered to intracellular resting levels. This suggests that vasopressin stimulation results in the activation of a phosphoinositidase C. It is probable that cortisol secretion by bovine ZFR cells in response to vasopressin is dependent upon activation of this Ca²⁺-independent phosphoinositidase C. However, the small magnitude of the cortisol secretory response makes it unlikely that vasopressin is a primary regulator of cortisol secretion in vivo.