Subchronic toxicity of 1,4-dichloro-2-nitrobenzene (DCNB) was examined by feeding F344 rats and BDF₁ mice of both sexes a diet containing 1,481, 2,222, 3,333, 5,000 or 7,500 ppm DCNB (w/w) for 13 wk. Oral administration of DCNB in feed to rats and mice retarded growth rates and induced subchronic toxicity affecting the liver, kidney, testes and blood. Liver and kidney were most responsive to DCNB. BMDL₁₀ values for relative liver weight were 12.0 and 22.6 mg/kg/d for male and female rats, respectively, and 88.7 and 94.4 mg/kg/d for male and female mice, respectively. Increased liver weights and centrilobular hypertrophy of hepatocytes were observed in the DCNB-fed rats and mice of both sexes. Both increased serum activities of AST and ALT and liver necrosis occurred in the DCNB-fed mice. Increased incidences of hyaline droplets and granular casts in the proximal renal tubules were observed only in the DCNB-fed male rats, indicating α_2υ-globulin nephropathy. Eosinophilic droplets in the renal tubular cells and increased BUN concentrations occurred in the DCNB-fed female rats. DCNB-induced testicular and hematologic changes were noted in rats and mice. On the basis of these results, the highest dose level for the 2-yr bioassay study of rodent carcinogenicity was determined to be 2,000 ppm.