Antenatal glucocorticoid treatment of mothers at risk of premature delivery is highly cost-effective in reducing neonatal mortality and morbidity. However, there is only limited information on the actual glucocorticoid bioactivity (GBA) reaching the fetus. By employing a recently developed recombinant cell bioassay, we studied circulating GBA in preterm newborns exposed to the standard antenatal betamethasone regimen (12 mg betamethasone twice, 24-h interval, for the mother; repeated in 7-10 d if required). Plasma GBA and cortisol concentrations were measured in cord blood of 71 infants (mean gestational age, 28.9 wk; range, 24.6-32 wk; mean birth weight, 1208 g; range, 480-2010 g). The median time between the last administered betamethasone dose and birth was 2.0 d. Cord GBA ranged from less than 15.6 to 170 nmol/liter cortisol equivalents. The level was highly dependent on the time between the last betamethasone dose and birth, i.e. infants born shortly (<12 h) after the last steroid dose displayed on average 4-fold higher GBA than that in the reference group (infants with >7 d since the last betamethasone dose before birth or without treatment; 74 vs. 21 nmol/liter cortisol equivalents; P < 0.0001). By contrast, if more than 72 h had elapsed between the last steroid dose and birth, circulating GBA was strongly dependent on cord cortisol (r = 0.85; P < 0.0001; n = 30). In multiple regression analysis adjusted for cord cortisol concentration and the time since the last steroid dose, increased umbilical artery resistance, a sign of severe fetal distress, was associated with lower cord GBA (P = 0.01). In conclusion, antenatal exposure of preterm fetuses to betamethasone causes a sizeable, but brief, peak of supraphysiological GBA, and approximately 3 d after the last betamethasone dose, circulating GBA derives from cord cortisol concentration.