The effects of gentamicin sulfate on carbonic anhydrase (CA) enzyme activity in in vitro human and in in vivo rat erythrocytes were investigated. For in vitro study, human carbonic anhydrase-I and -II (HCA-I and HCA-II) were purified by affinity-column chromatography, and rats were used for in vivo study. In vivo and in vitro CA enzyme activity was determined colorimetrically using the CO2-hydration method of Wilbur and Anderson as modified by Rickli et al. Gentamicin sulfate (1.98-9.90 mM) showed in vitro inhibitory effects on HCA-I and HCA-II hydratase activity up to a 2 mM concentration, when determined using the C02-hydratase method. Rat erythrocyte CA activity was significantly inhibited for up to 3 h (p<0.001) following intramuscular administration of gentamicin sulfate to Sprague-Dawley rats (3.2 mg/kg body weight). In conclusion, gentamicin sulfate inhibits CA enzyme activity in vivo and at low concentrations in vitro, but activated it at high concentrations (> or = 4 mM) in vitro.