The interaction of acetate ions with Mn(II) bovine carbonic anhydrase as was studied by NMR, revealed the close proximity between two anion molecules and the metal ion at the active center of the enzyme. However, only one acetate molecule is responsible for the inhibition of the enzymic activity. The two binding sites could also be distinguised by their ability to bind sulfonamide inhibitors. The distances between the acetate methyl groups and the Mn2+ was calculated using the longitudinal and transverse relaxation rates for the bound ligands, and were found to be 4.3 ± 0.3 and 4.8 ± 0.3 A ̊ for the acetate ions bound to the inhibitory and noninhibitory sites, respectively. These distances are consistent with a model in which the carboxylic groups of the acetate anion are directly coordinated to the metal. However, it is likely that at least the noninhibitory acetate, which is also untitratable by sulfonamide, is bound to another site.