Synthetic peptides corresponding to the region adjacent to His-94 in the small subunit of cytochrome b558 inhibited superoxide generation in a cell-free system. The shortest sequence that gave a half inhibitory concentration (IC50) lower than 50 microM was TRNYYVRAVL. Substitution of alanine for any of two tyrosine or central valine residues markedly increased IC50, thereby indicating that these residues are critical for the activity. Since the inhibition was observed when the peptide was added to the system before but not after the stimulation with sodium dodecylsulfate, the peptides seem to interact irreversibly with the cytochrome molecule and hinder electron transport or alternatively to interfere with the association between cytochrome b558 and cytosolic components.