Histidine-235 of human 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is the second basic residue in a conserved HXH motif. This residue is solvent accessible, readily reacting with the group specific reagent diethyl pyrocarbonate. Site-directed mutagenesis has been employed to substitute alanine or aspartate for H235. Characterization of the isolated H235A and H235D lyase mutants indicates that their tertiary structure is substantially intact. The mutant proteins, like the wild-type enzyme, are stoichiometrically modified by the affinity label, 2-butynoyl-CoA. Catalytic activity of the mutants is diminished by 15-fold and Km for HMG-CoA elevated approximately 4-fold in comparison with the values for wild-type enzyme. The function of H235 is suggested by investigation of the interaction of these enzymes with the dissociable divalent cation (e.g. Mg2+ or Mn2+) that is required for activity. ESR experiments show that wild-type enzyme forms a stable binary E*M complex. In contrast, H235A and H235D proteins do not efficiently form a binary complex. Significant interaction with cation (Mn2+) only occurs in the presence of the substrate analog, 3-hydroxyglutaryl-CoA. Similarly, when cation interaction is estimated in the presence of substrate using steady-state kinetic approaches, activator constants (Ka) and divalent cation Km values are measurable but are elevated by 15-90-fold over comparable estimates for the wild-type enzyme. The data confirm our earlier suggestion that both protein and substrate contribute ligands to HMG-CoA lyase's divalent cation activator. More specifically, the current observations suggest that H235 has an important function in cation binding.