The biliary excretion products recovered after iv administration of ethidium bromide to rats were found to consist of: unchanged drug, 8-acetylethidium, and an 8-acetylated hydroxylated metabolite. Proofs of structure were obtained by field desorption mass spectroscopy and 1H NMR, which indicated that the latter metabolite is probably substituted at position 2-. The yield of these various metabolites was critically influenced by pretreatment of the animals with inducers of the hepatic mixed function oxidases. Similar results were obtained in vitro, using various subfractions of differently pretreated rat liver. The role of each cellular fraction and order of completion of the acetylation and hydroxylation steps are determined and the putative occurrence of transient intermediates is discussed.