18-Hydroxylase activity, reported here for the first time in the mouse adrenal tumor cell line (Y1), was expressed in the metabolism of 11-deoxycorticosterone (DOC) and corticosterone (B). Detected after 24 h of incubation, it was more evident after 48 h and produced mostly 18-hydroxy-20 alpha-DHB from these exogenous substrates. However, 18-hydroxylation was quantitatively less significant than the metabolism of 20 alpha-reduction and 11 beta-hydroxylation (of DOC). The latter is also the predominant metabolism of progesterone in this cell line, during the conversion of cholesterol from the serum-supplemented culture media. The cytochrome P-450 11 beta activity of the Y1 cells is similar to that of the mouse in vivo which catalyzes the production of an 11 beta 18-dihydroxylated metabolite as the principal 18-hydroxylated steroid. It is different from that of other species, such as the rat and the bovine, both in terms of the ratio of 11 beta- to 18-hydroxylated metabolites and of the structure of these metabolites.