Transforming growth factor-β (TGF-β) is a peptide implicated in tissue injury and repair but its role in the premature human lung remains unclear. In the present study, we used a TGF-β responsive-promoter-luciferase construct in mink lung epithelial cells to quantify levels of biologically active TGF-β (BA-TGF-β) in the endotracheal aspirate (ETA) fluid from 16 extremely low birthweight neonates [6 M/10 F, mean GA 26 weeks (range 23–30), mean BW 774 g (range 555–1,075)]. ETA fluid was obtained on day 1 and then every 4 days up to 32 days. BA-TGF-β levels were low (92 ± 19 pg/ml) in the first 24 h of life and then increased 5- to 10-fold with peak BA-TGF-β levels (400 ± 50 pg/ml) on day 20–25. BA-TGF-β levels were higher in male than female infants (p = 0.0056). Prenatal steroids decreased significantly the amount of BA-TGF-β recovered. High initial levels of BA-TGF-β persisted over time and were predictive of the need for oxygen therapy at home. We conclude that abundant BA- TGF-β is present in the lungs of preterm infants and speculate that it may be involved in inflammatory and repair processes encountered in acute and chronic lung disease.