Recent in-vitro studies made by Askari, Monette & Leroux (1970), concerning the synthesis of C19 steroids by the adrenal gland of male and female rats, indicate that androstenedione, testosterone, dehydroepiandrosterone and 11β-hydroxyandrostenedione are metabolites of pregnenolone. It is generally accepted that 17αhydroxy-C21-steroid intermediates are required for side chain cleavage in the transformation of either pregnenolone or progesterone to C19 steroids (Savard, Dorfman, Baggett & Engel, 1956; Slaunwhite & Samuels, 1956); conflicting reports, however, have appeared regarding their formation in the rat adrenal gland (Bush, 1953; Roche, Michel & Jouan, 1959; Brownell, Lee, Beck & Besch, 1963; Laplante, Giroud & Stachenko, 1964; Karaboyas & Koritz, 1965; Young & Sweat, 1967). To provide supporting evidence for the 17α-hydroxy pathway, we attempted to isolate and identify some of the possible intermediates leading from pregnenolone to the isolated C19 metabolites. Adrenal gland tissue (2·0 g) from adult male