The acute toxicity of hydrocortisone 17-butyrate 21-propionate (HBP), hydrocortisone 17-butyrate (HB17) and hydrocortisone 21-butyrate (HB21) were investigated by three administration routes (s.c., i. p. and p. o.) in mice, rats and dogs. In the case of HBP, LD50 by oral administration was the highest, and followed by subcutaneous and intraperitoneal administration in mice and rats. And LD50 of HB17 and HB21 were not different from HBP in mice by subcutaneous administration. The depression of spontaneous movement and respiratory rate, ptosis, larcrymation and the collapse were commonly observed in all drugs, and it was independent of administration routes. The autopsy revealed the atrophy of thymus, spleen and adrenal glands, the supprative nodules of heart and liver and the ulcers of alimentary tract in mice and rats. But the changes observed in mice and rats were recognized when 1000 mg/kg of HBP was administered to dogs subcutaneously. Many of these changes were common to glucocorticoids, and the LD50 of HBP was rather high compared with other synthetic steroids; therefore, HBP was among less toxic steroid.