The soluble catalytic domain of NADH-cytochrome b5 reductase was radiolabeled with [14C]N-ethylmaleimide. Reaction for a limited time resulted in incorporation of 0.41 eq of N-ethylmaleimide and loss of 36% of the enzyme activity. Chromatography on a 5'-ADP affinity column separated the reductase which was modified with N-ethylmaleimide from the unreacted enzyme; the isolated derivative constituted 37% of the total material, was completely inactivated, and contained 1.00 eq of N-ethylmaleimide. Cyanogen bromide cleavage of the derivative demonstrated that radioactivity was limited to a single peptide which contained both Cys-283 and Cys-297. Tryptic hydrolysis of this cyanogen bromide peptide showed that the radioactivity was associated with Cys-283. Automated sequenator analysis confirmed that Cys-283 was the radiolabeled residue. These data demonstrate unambiguously that Cys-283 provides the essential thiol group of cytochrome b5 reductase.