The role of cysteine 266 in human 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, a residue that is homologous to a cysteine mapped to the active site of prokaryotic HMG-CoA lyase by protein chemistry approaches, has been investigated by site-directed mutagenesis. Both the wild-type human enzyme and a C323S variant, in which a regulatory sulfhydryl has been eliminated without any negative effect on catalytic activity (Roberts, J. R., Narasimhan, C., Hruz, P. W., Mitchell, G. A., and Miziorko, H. M. (1994) J. Biol. Chem. 269, 17841-17846), were used as models. Mutant enzymes C266A, C266A/C323S, C266S, and C266S/C323S were overexpressed in Escherichia coli and purified to homogeneity. In all cases, kinetic characterization indicated that the Km value for HMG-CoA was not substantially different from the value measured using wild-type human lyase, suggesting that no serious structural perturbation occurs upon replacing Cys-266. A dissociable divalent cation (Mn2+ or Mg2+), which is required for activity in both native and C323S enzymes, is also an essential component for activity in each of the Cys-266 mutants. The structural integrity of the human mutants was further indicated by Mn2+ binding studies, which demonstrate similarities not only in the activator cation binding stoichiometries, but also in the KD values for Mn2+ as determined for wild-type and mutant C266A or C266S proteins. Purified C266A and C266A/C323S mutants both displayed approximately 1.3 x 10(4)-fold diminution in specific activity, while the kcat value was diminished in both C266S and C266S/C323S by approximately 9.9 x 10(2)-fold. This large diminution in catalytic efficiency in enzyme variants that display no substantial structural perturbations is in accord with an active-site assignment to Cys-266 and qualifies its sulfhydryl group for consideration as a component of the catalytic apparatus.