Hepatocyte nuclear factor-3β (HNF-3β) is a member of the winged helix family of transcription factors that is thought to play a role in the formation of the embryonic foregut endoderm and in control of gene expression and differentiation of the respiratory epithelium. In order to further define a role of HNF-3β in lung development, the temporal-spatial distribution of HNF-3β was assessed by immunostaining with polyclonal antisera generated against rat HNF-3β (kindly provided by Dr. R. Costa) using avidin-biotin peroxidase staining after antigen retrieval. Fetal and postnatal human tissue from 21 fetuses from 10-23 weeks gestation, from 4 infants dying from non-pulmonary causes (36-42 weeks) and 24 infants with hyaline membrane disease (HMD) or bronchopulmonary dysplasia (BPD) from 2 days to 7 months of postnatal ages were assessed. HNF-3β was first detected in human fetal lung at 10 weeks gestation, where it was confined to the nuclei of respiratory epithelial cells in the trachea, bronchi and developing bronchioles and terminal airways. Thereafter, HNF-3β staining was detected in the respiratory epithelium of most infants, generally being expressed more widely in the distal vs. the proximal airways. In postnatal infants, without primary lung disease, staining was generally less intense than in fetal, and was confined to alveolar portals and Type II cells. In infants with BPD, HNF-3β staining was most intense in distal airways, most abundantly in the periphery in regions of regeneration. In the adult lung, HNF-3β was detected in only alveolar Type II cells. The temporal-spatial pattern of HNF-3β paralleled that of TTF-1 (thyroid transcription factor-1) and proSP-B. Of other tissues tested, HNF-3β was detected only in fetal pancreas, in the duodenal crypts of Lieberkühn and in the stomach epithelium. The thyroid was unstained. The temporal-spatial pattern of HNF-3β staining in the respiratory epithelium is consistent with its role in epithelial differentiation, regeneration and in surfactant protein gene expression.