A nitrate reductase (EC 1.6.6.1)-inactivating factor has been isolated from 8-day-old wheat leaves. The purification schedule involved ammonium sulfate precipitation, Sephadex G-100 filtration, DEAE-cellulose chromatography, and Sephadex G-150 filtration. No accurate assessment could be made as to the degree of purification relative to crude extract as the inactivating factor could not be detected in crude extract. However a 2,446-fold purification was achieved from the ammonium sulfate fraction to the pooled enzyme from the Sephadex G-150 step.The inactivating factor was heat-labile and had a molecular weight of 37,500. The inactivating factor was particularly sensitive to the divalent metal chelators, 1,10-phenanthroline and bathophenanthroline. Evidence indicated that Fe2" may be the functional metal. The trypsin inhibitors Na-p-tosyl-L-lysine chloromethyl ketone and a-N-benzoyl-L-arginine were inhibitory. However, phenylmethyl sulfonyl fluoride, an inhibitor of serine peptide hydrolases, was not inhibitory. Neither casein nor hemoglobin nor a range of artificial substrates were hydrolyzed by the inactivating factor.Highly purified wheat leaf nitrite reductase (EC 1.7.993) and ribulose 1,5-bisphosphate carboxylase:oxygenase (EC 4.1.139) were not affected by the nitrate reductase-inactivating factor.The inactivating factor was more active toward the NADH-nitrate reductase compared to either of the component enzymic activities flavin adenine mononucleotide-nitrate reductase and methyl viologen-nitrate reductase. The NADH-ferricyanide reductase (diaphorase) component was the least sensitive.In a previous paper (17) we reported on the occurrence in crude extracts from wheat leaves (Triticum aestivum L.) of two types of factors which appeared to affect the in vitro stability of highly purified nitrate reductase. One of these factors (II) reduced the stability of NR3 while the other factors (I and III) seemed to confer stability on NR.