The metabolism and irreversible protein binding of 2,4-[3,5-3H]dinitrofluorobenzene (3H-DNFB), a model chemically reactive compound, were studied in the rat. 3H-DNFB given intravenously (5 micrograms, 5 mg or 25 mg per kg) to anaesthetized cannulated rats was rapidly metabolized via the mercapturic acid pathway. The metabolites were extensively eliminated in bile and urine: predominantly as the glutathione conjugate and mercapturate in bile, and as the mercapturate in urine. Only ca. 3-10% of the doses remained in the liver, kidneys, spleen, heart and lungs at 3 hr. Dinitrophenyl mercapturate was the principal urinary metabolite in conscious rats dosed i.v. (5 mg or 25 mg per kg). Only 15-25% of the radiolabelled material in liver and kidney at 3 hr was irreversibly bound to protein, but 45-99% of that in the other organs and 49-88% in plasma was irreversibly bound. Preliminary evidence for the metabolism of 3H-DNFB (5 mg/kg and 25 mg/kg doses) to N2-acetyl-N6-DNP-lysine, a novel conjugate and metabolite of dinitrophenylated proteins in vivo, is presented.