The effects of oral doses of 2,4-dinitrotoluene (2,4-DNT) after oral administration for up to 24 months were studied in dogs, rats and mice. Ancillary studies included cytogenetics analysis in dogs and rats and three generation reproduction, dominant lethal mutation and metabolism studies in rats. In dogs, 0.2 mg/kg/day by capsule had no apparent effects, 1.5 mg/kg/day was toxic to some and 10 mg/kg/day was toxic to all and lethal to some. In rats, 0.57 or 0.71 mg/kg/day in feed (males or females) had no apparent effects, 3.9 or 5.1 mg/kg/day was toxic to some and 34 or 45 mg/kg/day was toxic to all and shortened lifespan. In mice, 13.5 mg/kg/day in feed was slightly toxic to some, 95 mg/kg/day toxic to all and 900 mg/kg/day halved lifespan. Target organs included the blood (methemoglobinemia with Heinz bodies and other sequelae), central nervous system (incoordination and paralysis), liver (hepatocellular carcinoma in rats), kidney (cystic tumors in mice) and gonads (decreased spermatogenesis in males of all three species; decreased corpora lutea in female mice). Pigment deposits (from metabolites and/or methemoglobin) were found in livers, kidneys and other organs, especially in mice. Rats had an increased incidence of the background subcutaneous and mammary tumors. No specific effects were seen in the ancillary studies (cytogenetics, dominant lethal mutation, reproduction, metabolism).