Brain factor-1 (BF-1) is a winged-helix transcription factor which has been shown to be essential for the development of the cerebral hemispheres. We report here the cloning and characterization of the mouse BF-1 gene. We have identified two classes of alternatively spliced mouse BF-1 cDNA which are transcribed from distinct promoters. Both classes of cDNA encode identical BF-1 proteins. The class 1 mouse cDNA is the homolog of the previously reported rat and human BF-1 cDNAs, and accounts for about 90–95% of BF-1 expression in brain. Primer extension analyses show that the major promoter, P1, is TATA-less and has multiple transcription start sites. We identified neural cell lines which express BF-1 primarily from the P1 promoter, including the OBL21a and OBL21 cell lines derived from the oldfactory bulb. Expression of BF-1 is highest in proliferating OBL21 cells, declining as the cells differentiate in vitro. This correlates well within the reduction of BF-1 expression as the cells of the telencephalic neuroepithelium differentiate. Using these cells, we demonstrate that the genomic region between −3.7 kb and −79 bp upstream of the P1 promoter contains cell-specific enhancer activity in transient transfection assays.