The existence and importance of the Na+/H+ exchanger in intracellular pH (pHi) regulation in ovarian cells was studied in acid-loaded avian granulosa cells by monitoring the recovery of normal pHi using a trapped fluorescein derivative as an indicator. The resting pHi of freshly isolated granulosa cells from preovulatory follicles was 680 ± 0·08 when the extracellular pH (pHo) and sodium concentration (Nao +) were 7·3 and 144 mmol/l respectively. While exposure of granulosa cells to high pHo (pHo > 745) medium shifted the pHi upward with time, incubation of the cells in low pHo (pH < 6·80) buffer resulted in a significant decrease in pHi. In contrast, pHi remained constant when pHo was varied between the broad range of 6·8–7·4. When the cytoplasm was acidified by treatment with nigericin in choline+ buffer, both the magnitude and rate of recovery of normal pHi was suppressed significantly with decreasing pHo, but increased in high pHo medium. The recovery of pHi was dependent upon the concentration of extracellular sodium, in that the recovery rate and magnitude increased concomitantly with increases in Nao + concentrations, while the recovery was abolished when Nao + was completely replaced with choline+. In addition, the sodium ionophore monensin enhanced the recovery rate of normal pHi in a concentration-dependent manner. This action of monensin was observed only when sodium was present in the incubation medium, indicating that Nao + entry is important for the recovery of normal pHi. Monensin also evoked further cytoplasmic alkalinization in fully recovered cells, with a relative net effect dependent upon the level of Nao + present. The recovery of pHi by acid-loaded cells was attenuated in a concentration-dependent manner by the Na+/H+ exchange inhibitor amiloride. These results clearly demonstrate in granulosa cells the presence of a pHi-regulating system that requires extracellular Na+ and is sensitive to amiloride.