The effects of acetylcholine (ACh) on intracellular free calcium were studied in primary cultures of purified bovine adrenal zona fasciculata/reticularis (ZFR) cells. In fura-2 loaded single cells, concentrations of ACh which stimulated cortisol secretion and phosphoinositol production were found to promote an increase in free cytosolic calcium ([Ca2+]i). This response was heterogeneous, showing either (i) an initial increase in [Ca2+]i followed by a fall to a level above that in unstimulated cells, or (ii) an initial increase followed by oscillations about the original resting level or a higher resting level. The frequencies of [Ca2+]i oscillations to ACh showed a dose-dependent trend. The sustained [Ca2+]i oscillations were abolished by the muscarinic antagonist atropine, or by removal of extracellular Ca2+. These data demonstrate for the first time in adrenocortical cells that: (i) ACh can induce [Ca2+]i oscillations in single ZFR cells; (ii) these oscillations occur in a dose-dependent manner; (iii) the sustained oscillatory phase is dependent on influx of extracellular Ca2+. Thus, like cells of the zona glomerulosa, bovine ZFR cells are also capable of sustained dose-dependent oscillatory responses to agonists which activate phosphoinositidase C.