Testosterone metabolism was studied in human adult and fetal liver microsomes. In fetal livers 6 beta-hydroxylase (6 beta OH) activity (1-2% of adult activity) and 2 alpha-hydroxylase (2 alpha OH) activity (about 40% of adult activity) were present. Also some fetal livers produced two unknown metabolites. Androstenedione was formed in all fetal livers studied (10-20% of adult activity). Testosterone hydroxylations at 6 beta-, 2 beta-, 15 alpha- and 15 beta-positions were associated with CYP3A isoform(s) in adult liver, because they were strongly inhibited by midazolam, a known substrate for CYP3A4 and by anti-CYP3A4 antibody. Fetal liver activities were consistently inhibited less than the activities in adult livers. The formation of androstenedione was not affected by these inhibitors in fetal or adult liver microsomes. Benzphetamine N-demethylase activity in the fetal livers was about 40% of adult activity. Anti-CYP3A4 antibody had no effect on that activity in fetal or in adult liver microsomes, whereas a monoclonal antibody 1-68-11 (generated against rat CYP2C11) slightly inhibited benzphetamine N-demethylase activity in adult liver. This study indicates that human fetal and adult liver are dissimilar in their testosterone metabolism pattern. The formation of androstenedione from testosterone in fetal liver may have a physiological role. Testosterone hydroxylases are less inhibited by anti-CYP3A4 antibody, midazolam and progesterone in fetal than in adult liver.