Bacterial luciferase [from Beneckea harveyi] is a heteropolymeric protein (.alpha..beta.) that catalyzes the conversion of chemical energy to light by oxidation of a FMNH2 and a long chain aliphatic aldehyde. Elucidation of the specific amino acid residues involved in the enzymatic reaction is essential for understanding the mechanism of the bioluminescent reaction. Luciferase was inactivated by ethoxyformic anhydride with a 2nd-order rate constant of 146 M-1 min-1 at pH 6.1 and 0.degree. C with a concomitant increase in absorbance at 240 nm due to formation of ethoxyformylhistidyl derivatives. Activity could be restored by hydroxylamine and the pH curve of inactivation indicated the involvement of a residue having a pKa of 6.8. Both substrates, FMNH2 and aldehyde, protected the enzyme against inactivation, suggesting that the modification occurred at or near the active site. Incorporation of [14C]ethoxyformyl groups into luciferase indicated that inactivation resulted from the modification of .apprx. 3 histidyl residues, 1 histidine being found on the .alpha. subunit and 2 on the .beta. subunit. Hybridization experiments, in which ethoxyformylluciferase, .alpha.m.beta.m, was complemented with native subunits, .alpha. or .beta., showed that the hybrid, .alpha.m.beta., has the same activity as .alpha.m.beta.m whereas the activity of the hybrid, .alpha..beta.m, was close to that of the reconstituted luciferase, .alpha..beta.. Modification of only 1 histidyl residue on the .alpha. subunit apparently inactivates luciferase, suggesting that this histidyl residue plays an essential role in the mechanism of the bacterial bioluminescent reaction.