Myogenic regulatory factors (MRFs) are hierarchical regulators of skeletal myogenesis. Many MRF promoters have been well characterized with respect to flanking sequences that control their expression. Yet the promoter elements that regulate Myf-5, the first MRF expressed during mammalian embryogenesis, are still largely unknown. Comparison of Myf-5 5' flanking regions from bovine, mouse, and chicken genes revealed three evolutionarily conserved elements proximal to the transcription start site: the TATA box, an octamer motif, termed OLS, and a 6-bp C-rich element. Mobility shift assays and DNase I footprinting analysis demonstrated that a nuclear factor(s) present in both bovine and avian muscle and nonmuscle tissues specifically recognized OLS. Furthermore, this binding activity reacted with a polyclonal Oct-1 antibody. In avian primary myoblast and fibroblast cultures, CAT reporter constructs under regulation of the proximal Myf-5 5' flanking sequence were expressed preferentially in myoblasts with CAT levels approximately 12-fold higher than in fibroblasts. The TATA box and octamer motif were important for expression in both myoblasts and fibroblasts: loss of the TATA box abolished activity, and disruption of the OLS resulted in 50-75% loss of promoter activity.