Pregnenolone (Preg) and 17-hydroxypregnenolone (17OH-Preg) are marker steroids that are elevated in the 3beta-hydroxysteroid-dehydrogenase-II deficiency form of congenital adrenal hyperplasia. The aim of this study was to establish normative data for both steroids in healthy preterm (28-33 and 34-37 wk gestation) and full-term infants, because reference values for the early neonatal period do not exist. At delivery, the main source of Preg is the placenta, because the highest levels were found in the retroplacental space (median, 141.31 nmol/liter), with no significant difference between preterm and full-term pregnancies. The fetal adrenals provide most of the circulating 17OH-Preg in full-term neonates, as demonstrated by a marked arteriovenous gradient in cord blood (40.96 nmol/liter vs. 10.77 nmol/liter). 17OH-Preg levels in the umbilical arteries were significantly lower in premature infants than in full-term infants (8.06 nmol/liter vs. 40.96 nmol/liter). During the first 2 postnatal weeks, Preg concentrations showed a rapid and significant fall in early preterm infants [95.78 nmol/liter (0 h) to 36.69 nmol/liter (d 14)] as well as in full-term infants [66.62 nmol/liter (0 h) to 14.81 nmol/liter (d 6)]. In addition, a significant fall in 17OH-Preg levels was found in full-term neonates [40.96 nmol/liter (0 h) to 11.32 nmol/liter (d 6)]. After 12 h, significantly higher levels for Preg and 17OH-Preg were found in early preterm infants (98.01 nmol/liter and 69.13 nmol/liter), compared with full-term neonates (36.29 nmol/liter and 28.55 nmol/liter, P < 0.05), reflecting the increased fetocortical activity as a response to the stress of delivery in the prematures. With these longitudinal data, it is now possible to confirm or exclude the diagnosis of 3beta-hydroxysteroid-dehydrogenase-II deficiency within the first postnatal week.