The inhibition of urease by heavy metal ions has been habitually ascribed to the reaction of the ions with enzyme thiol groups, resulting in the formation of mercaptides. To probe the modes of metal binding to the enzyme, in this work the reaction of mono- (Ag, Hg) and di- (Cu, Hg) valent metal ions with jack bean urease was studied. The enzyme was reacted with different concentrations of the metal ions for different periods of times, when its residual activity was assayed and thiol content titrated. The titration carried out with DTNB was done to examine the involvement of urease thiol groups in metal ion binding. The binding was further probed by reactivation of the metal ion-enzyme complexes with DTT, EDTA and dilution. The results are discussed in terms of the HSAB concept. In inhibiting urease the metal ions showed a common feature in that they inhibited the enzyme within a comparable micromolar range, and also in that their inhibition was multisite. By contrast, the main distinguishing feature in their action consisted of the involvement of enzyme thiol groups in the reaction. Hg (2+) and Hg2(2+) inhibition was found thoroughly governed by the reaction with the enzyme thiols, and the complete loss of enzyme activity involved all thiols available in the enzyme under non-denaturating conditions. In contrast, Ag+ and Cu2+ ions for the complete inactivation of the enzyme required 53 and 60% of thiols, respectively. Accordingly, Ag+ and Cu2+ binding to functional groups in urease other than thiols, i.e. N- and O-containing groups, cannot be excluded. Based on the reactivation experiments this seems particularly likely for Cu2+, whose concurrent binding to thiols and other groups might distort the architecture of the active site (the mechanism of which remains to be elucidated) resulting in the observed inhibitory effects.