The antithyroid acting drug propylthiouracil (PTU) was administered to male and female Wistar rats at 0, 0.1, 1, or 10mg/kg body weight for 4 weeks according to the draft protocol of the “Enhanced OECD Test Guideline 407” (enhanced TG 407) in order to investigate its suitability to detect endocrine-mediated effects. The study was conducted with two identical subsets of five animals per sex and dose each to provide data on sensitivity. The modified protocol includes the investigation of additional organ weights, pathology, and histopathology, of thyroid hormones, of spermatozoa, and of estrus cycle. At time of sacrifice, all females were in the diestrus stage as prescribed. Adverse effects were observed in the thyroid gland (hypertrophy/ hyperplasia) and the pituitary gland (hyperplasia of basophilic cells, hypoplasia of acidophilic cells) together with dose-related decreased serum triiodothyronine (T3) and thyroxine (T4) levels and increased thyroid stimulating hormone (TSH) levels. Other effects of PTU included decrease of organ weights, anaemia, impaired blood coagulation, and reduced activity of enzymes. Hence, some of the additional examined endpoints of the enhanced TG 407, e.g., examination of pituitary gland and thyroid hormones, were suitable to detect endocrine-modulating effects of propylthiouracil. Treatment of five animals provides sufficient sensitivity to detect the described adverse effects of propylthiouracil. The enhanced TG is currently under investigation in several laboratories, evaluation of all the results will allow determining its practicability as well as the most suitable additional endpoints.