Several hormone agonists exert their physiological actions by triggering an inositol phospholipid—Ca2+ signalling cascade and cytosolic alkalinization. Although calcium ionophores have been used extensively to probe the role of Ca2+ in the regulation of steroidogenesis in granulosa cells, the precise relationship between changes in intracellular Ca2+ (Ca2+ i) and pH (pHi) is unclear. In the present study we have used a fluorescent pH indicator, 2′7′-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein, to examine the influence of two Ca2+ ionophores, ionomycin and 4-Bromo-A23187 (4-Br-A23187), on pHi in chicken granulosa cells. Chicken granulosa cells from the largest preovulatory follicle were incubated with Ca2+ ionophores (0–2 μm) and/or inhibitors of Na+/H+ antiport (amiloride, dimethylamiloride and ethylisopropyl amiloride; 0·5, 5 and 50 μm respectively) in the presence of Na+ (or choline+; 0–144 mm) and/or Ca2+ (0–10 mm). Ionomycin or 4-Br-A23187 elicited a rapid and sustained cytosolic alkalinization. The magnitude of increase in pHi was dependent on the concentration of the Ca2+ ionophore and the presence of extracellular Ca2+ but independent of extracellular Na+. Pretreatment of the cells with amiloride or its analogues failed to affect the increase in pHi induced by the Ca2+ ionophores significantly. These findings demonstrate that, in addition to their widely reported effects on Ca2+ i redistribution in granulosa cells, 4-Br-A23187 and ionomycin cause Ca2+-dependent cytosolic alkalinization. This action of the Ca2+ ionophores is independent of the Na+/H+ antiport. Caution must be exercised in using Ca2+ ionophores as probes to define the role of Ca2+ in the regulation of granulosa cell function.