The presence of carbonic anhydrase (type V) was recently documented in rat and mouse pancreatic islet B-cells by immunostaining and Western blotting. In the present study, the activity of carbonic anhydrase was measured in rat islet homogenates. It was about four times lower than in rat parotid cells. The pattern for the inhibitory action of acetazolamide upon carbonic anhydrase activity also differed in islet and parotid cell homogenates, suggesting the presence of different isoenzymes. NaN₃ inhibited carbonic anhydrase activity in islet homogenates and both D-[U-¹⁴C]glucose oxidation and glucose-stimulated insulin secretion. Acetazolamide (0.3-10.0 mM) also decreased glucose-induced insulin output, but failed to affect adversely D-[U-¹⁴C]glucose oxidation, whilst inhibiting the conversion of D-[5-³H]glucose to ³HOH and that of D-[U-¹⁴C]glucose to acidic metabolites. Hydrochlorothiazide (3.0-10.0 mM), which also caused a concentration-related inhibition of the secretory response, like acetazolamide (5.0-10.0 mM), decreased H¹⁴CO₃^- production from D-[U-¹⁴C]glucose (16.7 mM). Acetazolamide (5.0 mM) did not affect the activity of volume-sensitive anion channels in B-cells, but lowered intracellular pH and adversely affected both the bioelectrical response to D-glucose and its effect upon the cytosolic concentration of Ca²⁺ in these cells. The lowering of cellular pH by acetazolamide, which could well be due to inhibition of carbonic anhydrase, might in turn account for inhibition of glycolysis. The perturbation of stimulus-secretion coupling in the B-cells exposed to acetazolamide may thus involve impaired circulation in the pyruvate-malate shuttle, altered mitochondrial Ca²⁺ accumulation, and perturbation of Cl^- fluxes, resulting in both decreased bioelectrical activity and insulin release.