OBJECTIVE To analyze hypothalamic-pituitary-adrenal axis balance in healthy newborns after antenatal betamethasone treatment for lung maturation where delivery could be prolonged until or near term. METHODS In a prospective observational study, salivary cortisol and cortisone levels were measured at the fourth day of life during resting conditions and in response to a pain-induced stress event in 23 neonates with antenatal exposure to a single course of betamethasone (2x12 mg) and compared with 40 controls. The mean interval between betamethasone treatment and delivery was 60+/-23 days. RESULTS On day 4 of life, neonates in the control group exhibited a significant increase in cortisol and cortisone from baseline levels after the stress induction (1.175-2.4 ng/mL for cortisol and 11.35-18.15 ng/mL for cortisone [both P<.05]), whereas, in betamethasone-exposed neonates, cortisol and cortisone stress response was not significantly different from baseline levels (1.39-1.6 ng/mL for cortisone [P=.76] and 14.8-17.1 ng/mL for cortisol [P=.69]). No influence of gestational age at betamethasone administration (P=.76) or gestational age at delivery (P=.71) on stress response patterns was observed in a multiple stepwise regression. CONCLUSIONS A single course of antenatal betamethasone treatment induces a suppression of stress reactivity in healthy newborns.