A lately discovered carbonic anhydrase (hCA, EC 4.2.1.1), the mitochondrial hCA VB, was cloned, expressed, and purified. Kinetic parameters proved it to be 3.37 times more effective than hCA VA as a catalyst for the physiological reaction, with kcat = 9.5 x 10(5) s(-1) and kcat/K(M) = 9.8 x 10(7) M(-1) s(-1), being second only to hCA II among the 16 isoforms presently known in humans. We investigated the inhibition of hCA VB with a library of sulfonamides/sulfamates, some of which are clinically used compounds. Benzenesulfonamides were ineffective inhibitors, whereas derivatives bearing 4-amino, 4-hydrazino, 4-methyl, 4-carboxy moieties or halogenated sulfanilamides were more effective (Ki's of 1.56-4.3 microM). Among the 10 clinically used compounds, acetazolamide, benzolamide, topiramate, and indisulam showed effective inhibitory activity (Ki's of 18-62 nM). Three compounds showed better activity against hCA VB over hCA II, among which were sulpiride and ethoxzolamide, which were 2 times more effective inhibitors of the mitochondrial over the cytosolic isozyme. hCA VB is a druggable target and some of its inhibitors may lead to the development of novel antiobesity therapies.