The aim of this study was to determine whether substrate concentration was of crucial importance in the formation of testosterone by the chick embryo testis. Testes from 15 to 18-day old chick embryos were cultured in medium 199 with dehydroepiandrosterone 1, 2, 6, 7-3H or androstenedione-4-14C added at different concentrations. Testosterone was identified and measured by crystallization to constant specific activity or by gas chromatography-mass spectrometry. The formation of testosterone could not be demonstrated at a substrate concentration of 76 nM. Concentrations in the micromolar range yielded measurable quantities of testosterone which increased to about 10% of the added substrate when the concentration was 70 microM. However, the capacity to form testosterone was shared by other organs such as the ovary or the mesonephros. These results should settle definitively the question concerning the formation of testosterone by the chick embryo testis. When exposed to high substrate concentrations, the testis can form testosterone, but under physiological conditions there is no such testosterone formation. This conclusion is in agreement with the absence of any physiological role of testosterone in the chick embryo.