To study the biosynthesis of 20 alpha-dihydropregnenolone-sulfate (20P5-S) and 20 alpha-dihydroprogesterone (20P4) in the feto-placental unit, human fetal liver and placenta were incubated with various radioactive precursors which included 14C-pregnenolone (P5), 14C-progesterone (P4), 14C-20 alpha-dihydropregnenolone (20P5) and 14C-20P5-S. In fetal liver, it was found that sulfokinase and 20 alpha-hydroxysteroid dehydrogenase (20HSD) activities were localized in 105,000xg supernatant obtained by a conventional differential centrifugation method. As cofactors, sulfokinase and 20HSD require ATP and NADPH, respectively. When P5 was incubated for varying periods with 105,000xg supernatant of liver homogenate, the precursor decreased with incubation time, whereas labeled 20P5-S increased steadily. A rapid increase in the 20P5 followed by declining amounts with further incubation occurred, suggesting a possible role of 20P5 as intermediates in 20P5-S formation from P5. When 20P-S was used as a precursor in the incubation with placental homogenate for different periods of time, rapid conversion to P4 was observed with a bell-shaped curve depicting 20P5 and 20P4 whereas the recovery of P5 remained low throughout the incubation period. These in vitro results indicate that the placental P4 and 20P4 might be synthesized, in part, from 20P5-S which is derived from fetal liver during pregnancy.