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Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea to produce ammonia and carbon dioxide These enzymes, which are found in fungi, bacteria, and plants, show very similar structures. Despite an abundance of urease in vegetal tissues, the physiological role of this enzyme in plants is still poorly understood. It has been previously described that ureases from the legumes jackbean ( Canavalia ensiformis) and soybean ( Glycine max) have insecticidal activity and antifungal properties. This work presents the physicochemical purification and characterization of a urease from cotton ( Gossypium hirsutum) seeds, the first description of this enzyme in Malvaceae. The urease content varied among different cotton cultivars. Cotton seed urease (98.3 kDa) displayed low ureolytic activity but exhibited potent antifungal properties at sub-micromolar concentrations against different phytopathogenic fungi. As described for other ureases, the antifungal effect of cotton urease persisted after treatment with an irreversible inhibitor of its enzyme activity. The data suggest an important role of these proteins in plant defense.