The tyrosyl residues of porcine mitochondrial L-malate:NAD+ oxidoreductase (EC 1.1.1.37) have been studied spectrophotometrically and using selective chemical modification with iodine and tetranitromethane. CNBr hydrolysis and Sephadex G-25, G-50 and G-75 chromatography produced a peptide which contained two tyrosines in the native and the nitrated molecules when the nitration took place in an NAD+-oxaloacetate solution. Nitration in the absence of the substrates caused the tyrosyl residues to disappear. Spectrophotometric titrations indicate that one of the 10 tyrosyl residues in mitochondrial L-malate:NAD+ oxidoreductase titrate abnormally, while iodination experiments suggest that two fast-reacting tyrosines are not involved in activity. Nitration and iodination experiments, in conjunction with CNBr-mapping, suggest that two of the four nitrated tyrosyl residues are necessary for biological action. Titration of the sulfhydryl groups with 4,4-bis-dimethylaminodiphenyl carbinol before and after nitration indicate that none of the cysteinyl residues were oxidized by the tetranitromethane, thus ruling out the loss of enzyme activity due to the thiol oxidation.