The in vitro and in vivo inhibitory effects of 5-(3alpha, 12alpha-dihydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide ( 1 ), 5-(3alpha, 7alpha, 12alpha-trihydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide ( 2 ), 5-(3alpha, 7alpha, 12alpha-triacetoxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide ( 3 ) and acetazolamide on rainbow trout ( Oncorhynchus mykiss ) (RT) erythrocyte carbonic anhydrase (CA) were investigated. The RT erythrocyte CA was obtained by affinity chromatography with a yield of 20.9%, a specific activity of 422.5 EU/mg protein and a purification of 222.4-fold. The purity of the enzyme was confirmed by SDS-PAGE. Inhibitory effects of the sulfonamides and acetazolamide on the RT erythrocyte CA were determined using the CO 2 -Hydratase method in vitro and in vivo studies. From in vitro studies, it was found that all the compounds inhibited CA. The obtained I 50 value for the sulfonamides ( 1 ), ( 2 ) and ( 3 ) and acetazolamide were 0.83, 0.049, 0.82 and 0.052 muM, respectively. From in vivo studies, it was observed that CA was inhibited by the sulfonamides ( 1 ), ( 2 ) and ( 3 ) and acetazolamide.