AbstractBecause of the inhibitory effect of testosterone on lympho‐epithelial nodule formation in the bursa of Fabricius, possibly through the suppression of alkaline phosphatase activity of the surrounding mesenchyme, there appears the possibility of a comparable inhibitory effect on the thymus.1. A single dose of one tenth milliliter (2.5 mg) of testosterone propionate in sesame oil (Schering) was injected into chick eggs between the first and fifth days of incubation, i.e., before and during the time of differentiation of the thymus from the epithelial primordia and before the appearance of lymphoblasts. The thymuses of 195 chicks including 50 controls were examined after 18 days of incubation; grossly and microscopically the thymus gland was relatively unaffected in all but a few chicks.Because the thymus appeared unaffected by testosterone as opposed to its complete inhibitory effect on the bursa of Fabricius; and because of the apparent relationship of testosterone‐labile alkaline phosphatase to epithelial nodule formation in the bursa, further investigations were carried out on thymic differentiation and the possible role, if any, of alkaline phosphatase activity in the surrounding mesenchyme.2. Chick embryos between 5–10 days of incubation were fixed in 80% ethanol or absolute acetone, embedded in paraffin, serially sectioned and exposed to either Gomori's glycerophosphate or Burstone's naphthol AS‐MX phosphate procedures for alkaline phosphatase activity. The mesenchyme surrounding the developing thymus showed either a negative or a negligible reaction for alkaline phosphatase activity following these procedures. In contrast, the thymic primordium did exhibit slight phosphatase activity which appeared to be localized in the reticular‐epithelial cells.The difference in demonstrable alkaline phosphatase activity of the capsule of the thymus and of the subepithelial mesenchyme of the bursa of Fabricius suggests a fundamental difference in the differentiation of the two organs in the embryo