The interaction of hydroxyurea with the cytosolic isozymes of carbonic anhydrase (CA), hCA I and hCA II has been investigated by means of kinetic and spectroscopic techniques. Hydroxyurea acts as a weak, non-competitive inhibitor of both isozymes, for the 4-nitrophenyl acetate esterase activity, with inhibition constants around 0.1 mM for both isozymes. The spectrum of the adduct of hydroxyurea with Co(II)-hCA II is similar to the spectra of tetrahedral adducts (such as those with sulfamide, acetazolamide or cyanamide), proving a direct interaction of the inhibitor molecule with the metal center of the enzyme, whose geometry remains tetrahedral. Based on the X-ray crystal structure of the adducts of hCA II with ureate and hydroxamate inhibitors, the hypothetical binding of hydroxyurea is proposed to be achieved in deprotonated state, with the nitrogen atom coordinated to Zn(II), and the OH group of the inhibitor making a hydrogen bond with Thr 199. This binding may be exploited for the design of both CA as well as matrix metalloproteinase (MMP) inhibitors, since hydroxyurea is the simplest compound incorporating a hydroxamate functionality in its molecule. Indeed, such inhibitors of the sulfonylated amino acid hydroxamate type have been generated, with potencies in the low nanomolar range for both type of enzymes, CAs and MMPs.