To assess the toxicity of N-phenyl-1-naphthylamine, Sprague Dawley rats were repeatedly administered with the chemical by oral gavage daily at doses of 0, 4, 20, 100, and 500 mg/kg/day for 28 days, followed by a 14-day recovery period. A significant decrease or decreasing trend of red blood cell counts, hemoglobin concentration, hematocrit, and mean corpuscular hemoglobin concentration and a significant increase in reticulocyte counts were observed at a dose of 500 mg/kg in both male and female rats. Increase in blood urea nitrogen and sodium levels was observed in male rats that received 500 mg/kg; increase in serum total protein, albumin, and calcium levels and in albumin/globulin ratio were observed in female rats that received 500 mg/kg. Increase in relative liver weight in female rats that received 100 mg/kg and increase in the absolute and relative liver weights in both male and female rats that received 500 mg/kg were observed; increases in the absolute and relative spleen weights and absolute kidney weight in female rats that received 500 mg/kg were observed. Hypertrophy of centrilobular hepatocyte and extramedullary hematopoiesis in the spleen were observed in both male and female rats at doses of 100 and 500 mg/kg. Renal tubular dilatation and papillary necrosis were observed in both male and female rats that received 500 mg/kg. These changes had the reversible trend in the recovery period. Based on these results, the no-observed-effect-level of N-phenyl-1-naphthylamine after a repeated daily oral administration for 28 days was determined to be 20 mg/kg/day for both sexes.