The fetal zone of the adrenal gland is known to persist after preterm birth, but there is uncertainty as to how long adrenal fetal zone steroid production continues and how it is regulated. The purpose of this study was to test two hypotheses. First, that the urinary excretion of 3beta-OH-5-ene steroids persists until term, and then declines, as it does in full-term infants. Second, that the persistence of the fetal zone is due to continuing ACTH stimulation. A longitudinal observational study was undertaken in 22 preterm infants of 24-31-wk gestation. Sequential measurements were made of urinary 3beta-OH-5-ene steroids (fetal zone steroid metabolites), plasma dehydroepiandrosterone sulfate (DHEAS), and ACTH. Excretion of urinary 3beta-OH-5-ene steroids was 1500-2000 microg kg(-1) d(-1), persisting until term, and declining abruptly at approximately 42 wk postconceptional age (PCA), to levels comparable to term infants at the same PCA. Median plasma ACTH levels rose from <7.6 pg mL(-1) at 25-wk PCA to 34.5 pg mL(-1) at 46-wk PCA. Urinary 3beta-OH-5-ene steroids were highest when ACTH levels were lowest, and were declining when ACTH was rising. In four infants given dexamethasone, urinary excretion of 3beta-OH-5-ene steroids and plasma DHEAS were not suppressed fully, when plasma ACTH and cortisol, and urinary cortisol metabolites were. These data suggest that ACTH is not the sole regulator of the adrenal fetal zone steroid synthesis and that involution of the fetal zone is related to gestation rather than birth.