Two tyrosine residues of mercuric reductase (MerA), Tyr-264 and Tyr-605, which were shown by the X-ray crystal structure to be involved in metal binding, were changed to phenylalanine residues by site-directed mutagenesis, both singly (Y264F, Y605F) and to form a double mutant (Y264,605F). The effect of these mutations on Hg(II) reduction activity varied. While MerA Y605F has a similar apparent Km to the wild-type enzyme and an apparent kcat reduced by 6-fold, MerA Y264F has an apparent Km 5-fold lower than the wild type and apparent kcat 160-fold lower. The double mutant MerA Y264,605F has the same apparent Km as MerA Y264F, but its apparent kcat was reduced by a further 7-fold. These results show that the roles of the two tyrosine residues are not equivalent and that Y264 is important for catalysis, possibly by destabilizing the binding of Hg(II) to the two ligating thiolates at the active site of MerA.