The first anion inhibition study of the mitochondrial human carbonic anhydrase (hCA, EC 4.2.1.1) isoform hCA VB is reported. Fluoride, chloride, bromide, iodide, cyanate, thiocyanate, cyanide, azide, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, sulfate, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid were compared as inhibitors of the two mitochondrial isozymes hCA VA and hCA VB. These enzymes are involved in biosynthetic reactions leading to fatty acid and Krebs cycle intermediates biosynthesis in addition to acting as catalysts for the interconversion of carbon dioxide and bicarbonate. The anion inhibition profiles of the two isoforms are dramatically different. The best hCA VB inhibitors were cyanate, thiocyanate, cyanide and hydrogensulfide (K(I)s of 80-76 microM) whereas the least effective ones were the halides (K(I)s of 11-72 mM), with the best inhibitor being fluoride and the least effective ones bromide and iodide. Whereas hCA VA is not sensitive to bicarbonate inhibition (K(I) of 82 mM) similarly to the cytosolic isoform hCA II, hCA VB is well inhibited by this anion, with a K(I) of 0.71 mM. Overall, hCA VB is more sensitive to anion inhibitors as compared to hCA VA. Such data support prior suggestions that the two mitochondrial isozymes play different physiological functions.