Previous studies in isolated turtle bladder have demonstrated that high concentrations of carbonic anhydrase (CA) inhibitors limit H+ transport (JH) by reducing the catalyzed rate of CO2 hydroxylation and also by inhibiting some other step in the acidification process. One possibility is that these inhibitors alter the energy substrate requirement for JH. Recent work has demonstrated that JH may be dependent in part on glucose oxidation via the pentose shunt (PS). The present study was undertaken to determine whether CA inhibitors exert a direct effect on PS metabolism by turtle bladder. Acetazolamide and benzolamide at concentrations of 5 X 10(-4) M significantly reduced the rate of 14CO2 evolution from [1-14C]- but not [6-14C]glucose after JH was abolished by an adverse electrochemical gradient for JH+. These changes are consistent with a reduction in PS metabolism. These same sulfonamides also reduced glucose-6-phosphate dehydrogenase (G-6-PD) activity in mucosal cell homogenates. Acetazolamide decreased the Vmax of G-6-PD but not the Km and, therefore, appears to be a noncompetitive inhibitor of G-6-PD with an estimated Ki of 10(-4) M. The t-butyl analogue of acetazolamide, CL 13850, which is without CA inhibitory activity, had no measurable effect on G-6-PD activity. Accordingly, it is suggested the sulfonamide CA inhibitors may reduce JH by two modes of action, inhibition of CA and inhibition of G-6-PD.