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Applied Photophysics stopped-flow instrument has been used for measuring the initial velocities for the CO2 hydration reaction catalyzed by different CA isozymes, by following the change in absorbance of a pH indicator.Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5) as buffer, 0.1 M Na2SO4 (for maintaining constant the ionic strength), following the CA-catalyzed CO2 hydration reaction for a period of 10–100 s.Saturated CO2 solutions in water at 20 C were used as the substrate. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the inhibition constants. For each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. The kinetic constants kcat and kcat/Km were obtained by non-linear least-squares methods using PRISM 3.Stock solutions of inhibitors (5-Amino-1,3,4-thiadiazole-2-thiol, compound 2) were prepared at a concentration of 1–3 mM (in DMSO–water 1:1, v/v) and dilutions up to 0.01 nM done with the assay buffer.