Gd(III)-sulfonamide complexes incorporating macrocyclic rings of the DOTA/TETA type have been prepared and assayed for the inhibition of the metalloenzyme carbonic anhydrase (CA, EC 188.8.131.52). The cytosolic isoform, CA I, was poorly inhibited, whereas cytosolic CA II and transmembrane, tumor-associated CA IX and XII were inhibited in the low nanomolar range by the Gd(III) complexes. Magnetic susceptibility and relaxivity measurements proved that the Gd(III) complexes have comparable parameters to those of clinically used MRI contrast agents like Dotarem, Prohance and Omniscan in aqueous solution. Some Gd(III) complexes were investigated for the inhibition of extracellular tumor acidification in two cell lines overexpressing CA IX, the colorectal HT-29 cell line and the cervical HeLa carcinoma cell line. In both tumor types, a slight but significant reduction of tumor acidosis was detected. Gd(III)-sulfonamide conjugates may thus be of interest for developing imaging techniques or novel treatment strategies for the management of hypoxic tumors overexpressing extracellular CA isozymes such as CA IX and XII.