ACTH stimulation tests were performed in 15 premature infants in a serial fashion at the ages of 5--10 days and 27--31 days. Six of the 15 had subsequent ACTH stimulation tests at 6--8 weeks and/or 12--13 weeks. The pre- and post-ACTH serum cortisol and dehydroepiandrosterone sulfate (DHAS) levels were determined by radioimmunoassay. The mean basal levels of cortisol, 55 ng/ml, and DHAS, 4108 ng/ml, were significantly higher (P less than 0.05 and P less than 0.025, respectively) at 5--10 days than those of 24 ng/ml and 1858 ng/ml, respectively, at 27--31 days. The mean net change (delta) of cortisol after ACTH at 5--10 days, 95 ng/ml, increased significantly to 148 ng/ml at 27--31 days. However, deltaDHAS did not differ significantly between the two periods (1514 ng/ml vs. 972 ng/ml). Therefore, deltaDHAS/deltacortisol was lower (P less than 0.05) at 27--31 days than at 5--10 days. No further significant changes were observed after 4 weeks of age in the levels of the two steroids. There was little correlation of basal levels between cortisol and DHAS, nor between deltacortisol and deltaDHAS at any age period we studied. There were four infants whose mothers had prenatal steroid treatment for the prevention of hyaline membrane disease and their values were not different from the other infants.