AbstractRubratoxin B is a substituted higher homolog of byssochlamic acid in which the ethyl group is replaced by a six‐membered ring containing an unsaturated lactone. Hydrogenation of the unsaturated lactone greatly reduces the acute toxixity of the compound. The acute median lethal doses of rubratoxin B and hydrogenated rubratoxin in male mice were 5.6 and 31 mg/kg, respectively. Pretreatment with phenobarbital (60 rag/kg × 4 days) or with SKF 525A (40 mg/kg) failed to significantly alter the toxicity of either compound. Histopathological examination of mice treated with rubratoxin B (3.5 mg/kg) revealed areas of hepatic zonal necrosis and marked necrosis of kidney tubules. No necrosis was observed in mice treated with hydrogenated rubratoxin (20 mg/kg). Hepatic microsomal mixed function oxidase activity was significantly reduced in mice pretreated with rubratoxin B (0.5 mg/kg × 4 days), while pretreatment with hydrogenated rubratoxin (2.5 mg/kg × 4 days) failed to depress microsomal activity. Our studies document the requirement of an alpha, beta unsaturated lactone ring for rubratoxin B toxicity. Based on the results from these studies the mechanism for rubratoxin B induced toxicity is postulated to be due to its electrophilic properties and subsequent alkylating acti