Pharmacological studies have suggested that both Na:H and HCO₃:Cl exchangers regulate cytosolic pH (pH_i) in b-cells, but the role of each exchanger is uncertain. Having identified NHE1 as the major isoform of the Na:H exchanger expressed in islets, we used mice bearing an inactivating mutation of NHE1 (Swe mice - Cell 91: 139-148, 1997) to investigate the possible influence of pH_i on stimulus-secretion coupling. Islets from Swe mice and their control littermates were cultured overnight in RPMI medium containing 10 mM glucose. All experiments were then performed in HCO₃:CO₂ and Hepes:O₂ buffers. pH_i and cytosolic Ca²⁺ ([Ca²⁺]_i) were measured by the BCECF and FURA-PE3 methods, and insulin secretion (IS) was studied with perifused islets. Using the NH₄Cl pulse technique, we confirmed that Na:H and HCO₃:Cl exchangers participate in pH_i regulation and established that the lack of NHE1 is not compensated for by another Na:H exchanger in Swe islets. Stimulation with 15 mM glucose produced a similar increase of pH_i in control and Swe islets in HCO₃ buffer, and no change or a decrease of pH_i in Hepes buffer. In HCO₃ buffer, 15 mM glucose induced biphasic increases in [Ca²⁺]_i and IS in control and Swe islets. Similar increases were observed in Hepes buffer although pH_i was lower. The stimulation of IS by glucose is thus independent of an increase in pH_i and of the presence of NHE1. When the islets were depolarized by KCl in the presence of diazoxide, [Ca²⁺]_i was stably elevated, and glucose increased IS without further elevating [Ca²⁺]_i. In both control and Swe islets, this amplification of IS (augmentation of the action of [Ca²⁺]_c on exocytosis) occurred without a change in pH_i. The amplification of insulin secretion by glucose is thus also independent of an increase in pH_i. In conclusion, our results indicate that neither the Na:H exchanger nor the small changes in pH_i produced by glucose are involved in b-cell stimulus-secretion coupling.