The properties of human fetal liver (HFL) estrogen 16 alpha-hydroxylase (16 alpha-OHase) were studied in microsomal preparations and hepatocytes maintained in culture. A specific assay was developed for the determination of estrogen 16 alpha-OHase activity based on the enzymatic release of tritium from the C-16 alpha position of stereospecifically labeled 16 alpha-3H-labeled C18-steroids, viz. 17 beta-[16 alpha-3H]estradiol, 17 beta-[16 alpha-3H]estradiol 3-sulfate, [16 alpha-3H]estrone, and [16 alpha-3H]estrone sulfate. The percentage of tritium at the C-16 alpha position of 17 beta-[16 alpha-3H]estradiol was 92.5%. There was no kinetic isotope effect in the 16 alpha-hydroxylation of 17 beta-[16 alpha-3H]estradiol. HFL hepatocyte and microsomal 16 alpha-OHase activity was linear with incubation time for at least 2 h, with a hepatocyte number up to 6.7 X 10(6) cells/ml and a microsomal protein concentration up to 1 mg/ml. The apparent Km of 16 alpha-OHase for estrone sulfate (E1S) was greater than that for either 17 beta-estradiol (E2) or estrone (E1; 2.9-6.4 microM vs. 0.70-0.84 microM). The maximum velocity of HFL 16 alpha-OHase also was greater with E1S or 17 beta-estradiol 3-sulfate than with either E1 or E2, and E1S was the most efficient substrate. The apparent temperature optimum for the microsomal enzyme was 37 C, and the apparent pH optimum was 7.0. 16 alpha-Hydroxylation of E1S by HFL microsomes was inhibited noncompetitively by E1. The biosynthesis of estriol from E2 by fetal liver microsomes does not require an intermediate oxidation step of E2 to E1 as appears to be the case in vivo in the human adult; this was demonstrated by the formation of [17 alpha-3H]estriol from [17 alpha-3H] E2 in incubations with HFL microsomes. A number of growth factors, hormones, and xenobiotics were preincubated with hepatocytes for 24 or 72 h to test for stimulation/inhibition of 16 alpha-OHase activity. 16 alpha-OHase activity was stimulated by dexamethasone, forskolin, (Bu)2cAMP, cholera toxin, 1,2-benzanthracene, and phenobarbital. Diethylstilbestrol, E2, and progesterone did not alter hepatocyte 16 alpha-OHase activity, except when very high concentrations of E2 and progesterone were used, when they became inhibitory; other hormones and growth factors did not alter the basal levels of the enzyme.