The alcohol dehydrogenase (ADH) inactivator from aerobically grown rice (Oryza sativa) coleoptiles was shown to be associated with membranes which were recovered in sucrose gradients at peak density 1.13 grams per cubic centimeter. When Mg(2+) was included in the gradient, the inactivator was recovered at peak density 1.16 grams per cubic centimeter coinciding with the marker enzyme for endoplasmic reticulum, antimycin A-insensitive NADH cytochrome c reductase. ADH was recovered exclusively in cytosol fractions. The inactivator attacks ADH from several plant sources and from yeast. There was no evidence for proteolysis when pure yeast ADH was inactivated by the inactivator, but there was a loss of SH groups from ADH during inactivation which was restored after incubation with dithiothreitol under denaturing conditions. The inactivator did not attack other SH enzymes tested but did result in loss of SH groups from glutathione and dithiothreitol which prevent ADH inactivation. When O(2) was removed from the inactivator assay medium, the inactivation as well as the loss of SH groups from yeast ADH was significantly depressed.