The hereditary breast cancer susceptibility gene, BRCA2, is considered to be a tumor suppressor gene that may be involved in the cellular response to DNA damage. The transcript for this gene is cell cycle regulated with mRNA levels reaching a peak just before the onset of DNA synthesis. In order to define the mechanisms by which BRCA2 is transcriptionally regulated, we have begun to study upstream regulatory sequences. In this report, we define a minimal promoter region that has strong activity in human breast epithelial cells. Deletions of this sequence narrowed the strong basal activity to a region extending from -66 to +129 with respect to the BRCA2 transcriptional start site. This sequence demonstrated cell cycle regulated activity with kinetics similar to the endogenous transcript. Examination of the sequence revealed several consensus binding sites for transcription factors including an E-box, E2F and Ets recognition motifs. Electrohoretic mobility shift assays revealed specific protein binding to two sequences upstream of the start site; the palindromic E-box and an Ets/E2F site. Site-directed mutagenesis of either of these sites reduced both the basal activity in log phase cells and the cell cycle regulated activity of the promoter. Mutational inactivation of both sites within the same construct effectively eliminated promoter activity. Antibodies to candidate transcription factors used in super shift experiments revealed specific interactions between the BRCA2 promoter and the basic region/helix - loop - helix containing USF-1 and 2 proteins and Elf-1, an Ets domain protein. Binding of these factors depended upon the presence of intact recognition sequences. The USF factors were shown to bind predominantly as a heterodimeric complex of USF-1 and 2 while Elf-1 bound the promoter when it was not occupied by USF. Co-transfection studies with USF proteins and the varicella zoster IE62 protein provide evidence for the involvement of endogenous and exogenous USF in the activation of the BRCA2 promoter. We propose that interactions between USF-1, USF-2 and Elf-1 play an important role in the transcriptional regulation of the BRCA2 gene.