Plasma cortisol concentrations are known to be relatively low at birth in premature infants and to be suppressed by antenatal betamethasone therapy. It has been suggested that low cortisol levels in the newborn period reflect adrenal insufficiency and are associated with respiratory disease. To further examine this relationship we determined plasma cortisol levels during the first 2 weeks of life in infants ≤32 wk gestation enrolled in an ongoing, blinded multi-center trial of antenatal thyrotropin releasing hormone (TRH). Women in premature labor at ≤30 wk gestation received betamethasone and either TRH or placebo and clinical data were obtained on the infants. Blood was collected at birth (cord) and postnatally from 145 infants ≤32 wk gestation at 8 participating sites and cortisol was assayed by immunoassay using enhanced chemiluminescence (Nichols Inst.). Natural log values for the data were used to evaluate correlations with lung disease. The geometric mean cortisol concentration in cord blood was 2.4 μg/dl (2.3 and 2.8 μg/dl for male and female infants, respectively), and there was a significant positive correlation with gestational age. Cortisol levels increased after birth with levels of 6.5, 11.2, 8.5, 8.2 and 6.4 μg/dl at 2 h and 1, 3, 7, and 14 days of age, respectively. Cortisol concentrations were significantly higher in infants with RDS at 2 h, 1 day and 3 days but not in cord blood(maximal levels on day 1, mean 13.6 vs 7.6 μg/dl, p=0.003). Levels were not significantly different at all time points for infants with and without chronic lung disease (CLD) at 28 days (ongoing requirement for oxygen or ventilator), CLD at 36 wk postconceptional age, or adverse outcome (death or CLD) at 36 wk postconceptional age. We conclude that very low birth weight infants exposed to betamethasone treatment in utero increase their plasma cortisol after birth and in response to the stress of RDS; low postnatal cortisol concentrations do not appear to be a predictor of CLD.