Extracts from 1–3 day-old infant urine which had been hydrolysed by Helix pomatia enzyme followed by solvolysis, were subjected to thin-layer chromatography. A band of material giving the 3β-hydroxy- Δ 5 steroid colour reaction with antimony trichloride staining reagent and previously named “U 2”, was purified by further thin-layer chromatography and identified as 16β-hydroxydehydroepiandrosterone. Proof of identity was obtained by comparing the chemical and physical characteristics of the chemically synthesized compound, with those of the unknown. Techniques used were: thin-layer and paper chromatography in 5 systems, gas chromatography, mass spectroscopy, infrared spectroscopy and high temperature catalytic reduction. It is excreted in infant urine mainly as a diconjugate.