Transcription of the murine D7Rp2e gene is highly variable among species of the genus Mus, indicating that extensive modifications in the gene's regulatory elements have occurred during evolution. Since promoter regions are well known to harbor cis-acting information that controls gene transcription, we compared the sequence and function of the D7Rp2e promoter in several Mus species, with the goal of understanding the molecular mechanisms underlying the interspecies variations in expression. Three overlapping binding sites for nuclear factors (sites A, B, and C in proximal to distal order) were identified about 300 bp upstream of the transcriptional start site. The sequences of these sites differ between the species Mus domesticus and M. pahari, which exhibit distinct D7Rp2e expression phenotypes. Site A binds a factor called RPBF-I; sites B and C bind a distinct factor that is termed RPBF-II and is likely a member of the NF-I family of transcription factors. DNase I footprinting experiments with the M. domesticus promoter show that binding of RPBF-II at site B is very strong, while binding of RPBF-I and RPBF-II at sites A and C, respectively, is weak; in contrast, with the M. pahari promoter, factor binding at sites A and C is strong, while that at site B is weak. These differences in patterns of binding-site occupancy derive from changes in the affinities of individual sites for their cognate nuclear factors. Transient transfection experiments indicate that the M. pahari binding pattern is capable of repressing transcription of a linked reporter. Such repression may contribute to the differences in D7Rp2e expression between the two species. We suggest that the species-specific footprinting patterns represent the existence of a dynamic equilibrium between two states of nuclear factor binding, the nature of which can be modified during evolution to result in new patterns of gene transcription.