Concentrations of pregnenolone (Δ5P), dehydroepiandrosterone (DHEA), 16α-hydroxydehydroepiandrosterone (16α-OH DHEA), pregnenolone sulfate (Δ5P-S), and dehydroepiandrosterone sulfate (DHEA-S) were measured simultaneously by radioimmunoassay in individual, paired umbilical artery (UA) and vein (UV) sera from 18 normal term pregnancies, 6 in labor, 12 not in labor. Mean UA and UV levels ± SEM (ng/ml) were for Δ5P: 30.39 ± 1.69, 35.55 ± 3.06; DHEA: 12.31 ± 2.34, 3.66 ± 0.38; 16α-OH DHEA: 7.48 ± 0.63, 10.59 ± 0.78; Δ5P-S: 1,652 ± 154,1,486 ± 130; DHEA-S: 2,122 ± 134, ± 134,1,906 ± 134. Umbilical artery Δ5P-S, DHEA-S, and DHEA levels were significantly higher than UV levels, whereas the reverse was true for Δ5P and 16α-OH DHEA. The inverse arterio-venous (A-V) gradient for 16α-OH DHEA was contrary to previous published reports using pooled samples. Comparison by linear regression of paired UA and UV steroid concentrations of Δ5P, Δ5P-S, DHEA, and DHEA-S revealed a significant correlation (P < 0.01) for each steroid. Labor was associated with a significant increase in UA levels of DHEA-S and a smaller, but not quite significant, increase in UA levels of δ5P-S, while similar changes for unconjugated δ5-3β-hydroxysteroids were not observed. Mean A-V gradients between the group of patients in labor and those not in labor were not significantly different. These data demonstrate that: 1) a significant difference between UA and UV concentrations exists for A5P, DHEA, 16α-OH DHEA, δ5P-S, and DHEA-S; 2) there is a significant correlation between UA and UV concentrations for δ5P, DHEA, δ5P-S, and DHEA-S, implying that each fetoplacental unit maintains an equilibrium relative to these steroid concentrations in the umbilical circulation; 3) labor is associated with a significant increase in UA levels of DHEA-S and probably of δ5P-S.