In vitro inactivation of Neurospora crassa nitrite reductase (NAD(P)H: nitrite oxidoreductase, EC 1.6.6.4) can be obtained by preincubation of the enzyme with reduced pyridine nucleotide plus FAD. The presence of nitrite or hydroxylamine, electron acceptors for the N. crassa nitrite reductase, or cyanide, sulfite or arsenite, competitive inhibitors with respect to nitrite of this enzyme, protects the enzyme against this inactivation. Anaerobic experiments reveal that oxygen is required in order to obtain complete inactivation of nitrite reductase by preincubation with reduced pyridine nucleotide plus FAD. Also, inactivation is prevented if catalase is included in the preincubation mixture. The presence of hydrogen peroxide in the preincubation mixture increases the sensitivity of nitrite reductase to the in vitro FAD-dependent NAD(P)H inactivation. Neither electron acceptors, competitive inhibitors nor catalase, agents which protect the enzyme against the FAD-dependent NAD(P)H inactivation, can reverse this process once it has occurred.