Electrostatic interaction between NADH-cytochrome b 5 reductase and cytochrome b 5 was studied by site-directed mutagenesis. The target residues for mutagenesis were selected on the basis of the previously reported chemical cross-linking study of these two proteins, which implicated possible charge-pair interactions between Lys-41, Lys-125, Lys-162, and Lys-163 of the enzyme, and Glu-47, Glu-48, Glu-52, Glu-60, Asp-64 (group A), and heme propionate of cytochrome b 5 . Mutant reductases that lost one of the above-listed Lys residues showed higher K m values for cytochrome b 5 and lower k c a t values than those of the wild type, suggesting that all of the examined Lys residues participate in binding with cytochrome b 5 as reported previously. In contrast, a removal of one of (or even all of) the group A residues from cytochrome b 5 by mutagenesis caused no significant effect on the catalytic properties of cytochrome b 5 . Additional elimination of another set of negative residues (Glu-41, Glu-42, Asp-57, and Glu-63 (group B)), which are also located close to heme, elevated the K m value by more than five folds. These results suggest that there should be other acidic residue(s) than group A in cytochrome b 5 which participate in binding with NADH-cytochrome b 5 reductase.