Expression of the rabbit pulmonary surfactant protein A (SP-A) gene is lung-specific, occurs primarily in type II cells, and is developmentally regulated. We previously identified two E-box-like enhancers, termed the distal binding element (DBE) and proximal binding element (PBE), in the 5'-flanking region of the rabbit SP-A gene. In the present study, the PBE was used to screen a rabbit fetal lung cDNA expression library; a cDNA insert was isolated which is highly similar in sequence to human upstream stimulatory factor 1 (hUSF1). By use of reverse transcription polymerase chain reaction, two isoforms of rabbit USF1 (rUSF1) mRNAs were identified in fetal rabbit lung and other tissues. The levels of rUSF1 mRNAs reach a peak in fetal rabbit lung at 23 days gestation, in concert with the time of initiation of SP-A gene transcription. Binding complexes of nuclear proteins obtained from fetal rabbit lung tissue and isolated type II cells with the DBE and PBE were supershifted by the addition of anti-rUSF1 IgG. Binding activity was enriched in type II cells compared with lung fibroblasts. Overexpression of rUSF1s in A549 adenocarcinoma cells positively regulated SP-A promoter activity of cotransfected reporter gene constructs. It is suggested that rUSF1s, which bind to two E-box elements in the SP-A gene 5'-flanking region, may serve a key role in the regulation of SP-A gene expression in pulmonary type II cells.