3β,17β-Dihydroxy-androst-5-en-16-one-4-14C (16-oxo-Δ5-DIOL) has been biosynthesized and four placentas were perfused in situ at midpregnancy with tracer amounts of this compound in combination with dehydroepiandrosterone-7α-3H. Exclusively 3H-labelled oestrone (OE1) and 17β-oestradiol (OE2) and exclusively 14C-labelled 17β-hydroxy-androst-4-ene-3,16-dione (16-oxo-T), 3,17β-dihydroxy-oestra-1,3,5(10)-trien-16-one (16-oxo-OE2) and oestra-1,3,5(10)-triene-3,16β,17β-triol (16-epi-OE3) were isolated in a radiochemically homogeneous form from the placentas as well as from the perfusates. In addition, 16α,17β-dihydroxy-androst-4-en-3-one (16α-HO-T) and oestriol (OE3) were isolated from the placentas, but not from the perfusates. Among the 16-substituted compounds isolated, 16-oxo-OE2 was the quantitatively most important one in the placentas and 16-oxo-T in the perfusates. Crystallisation with authentic carrier indicated the absence of any androst-5-ene-3β,16α,17β-triol (Δ5-TRIOL), 3β,16α-dihydroxy-androst-5-en-17-one (16α-HO-DHA), 16β,17β-dihydroxy-androst-4-en-3-one (16β-HO-T) and 3,16α-dihydroxy-oestra-1,3,5(10)-trien-17-one (16α-HO-OE1) in the combined extracts of placentas and perfusates. In addition, chromatographic evidence indicated the lack of androst-5-ene-3β,16β,17β-triol (16-epi-Δ5-TRIOL) and 16α-hydroxy-androst-4-en-3,17-dione (16α-HO-A) in these sources. From the urine specimens 3H-labelled OE1, OE2 and 16α-HO-OE1 and both 3H and 14C-labelled 16-oxo-OE2, OE3 and 16-epi-OE3 were isolated. The 3H/14C ratio of urinary 16-oxo-OE2 was significantly higher than that of OE3 whereas the isotopic ratio of urinary 16-epi-OE3 was significantly lower than those of 16-oxo-OE2 and OE3. A concept is presented describing the formation mechanism of 16-oxo-OE2. OE3 and 16-epi-OE3 in the placental compartment of the human foeto-placental unit at midpregnancy.