We studied the hematological effects of single and repeated exposure to 1,3,5‐trinitroben‐zene (TNB) in rats. Male F‐344 rats were gavaged with TNB at 35.5 and 71 mg/kg in corn oil. Blood was collected 5 h and 24 h after a single oral dose or 24 h after daily oral doses for 4 or 10 d in four different set of experiments. A dose‐dependent methemoglobinemia was present only in blood collected 5 h after a single dose. A highly significant dose‐dependent anemia with reduced red cells, hemoglobin, and hemotocrit was present in rats receiving TNB (or 4 or 10 d. A dose‐dependent decrease in serum triglycerides was present in rats receiving TNB for 10 d. There was no hemolysis when rat erythrocytes were incubated with TNB (in vitro) for 9 h. Spectral changes of hemoglobin recorded during the incubation with TNB confirm methemoglobin formation and progressive denaturation of hemoglobin‐forming hemichromes. The significance of methemoglobin and hemichrome formation is discussed, and a probable hypothesis for the hemolytic anemia is suggested.