Regulation of intracellular pH is an essential function and may be especially significant in the B-cell in which the influence of glucose on electrical activity is modulated by alterations in pH. Two possible regulatory processes have been examined: Na/H and HCO3/Cl exchange, by using inhibitors, an ionophore, and changes of ionic concentrations. In the presence of 11.1 mM glucose we found that DIDS, an inhibitor of anion exchange, elicited a dose-response increase in the relative duration of the active phase with an ED50 of 99 microM. Probenecid (0.5 mM), an inhibitor of anion fluxes, also augmented the electrical activity (EA) due to glucose. Withdrawal of HCO-3 elicited constant spike activity followed by a resumption of burst activity with a greater duration of the active phase compared to control. These data are consistent with predicted cellular acidification. However, reduction of Cl-o by isethionate substitution produced no marked effect on EA. In contrast, SO-4- substitution for Cl- resulted in variable effects characterized by constant spike activity or a decrease in the duration of the active and silent phases along with silent hyperpolarization. Tributyltin, a Cl/OH, ionophore enhanced EA at 0.25 microM with 120 mM Cl-o, but reduced EA with 10 mM Cl- as would be predicted with either cellular acidification or alkalinization, respectively. Amiloride at 100 microM elicited constant spike activity perhaps due to inhibition of Na/H exchange. Reduction of Na+o from 142.8 to 40.8 mM had a similar effect and enhanced the influence of amiloride. It appears therefore that interference with putative pH regulatory mechanisms in the B-cell are consistent with the hypothesis that cell pH is involved in regulation of EA.