Skeletal myoblast cell proliferation and subsequent differentiation are dependent on developmentally regulated expression of the fibroblast growth factor receptor 1 (FGFR1) gene. We have previously reported the isolation and initial characterization of the chicken FGFR1 gene (cek1) promoter. Both distal and proximal regions of the promoter were identified as necessary for developmentally regulated transcriptional activity in proliferating myoblasts, including its down-regulation in differentiated muscle fibers in vitro. Here we report detailed characterization of the molecular mechanism regulating FGFR1 promoter activity via the distal promoter region in proliferating myoblasts. This region was identified as a 242 base pair segment located greater than 1 kilobase upstream from the start of transcription that conferred increased transcriptional activity to a minimal thymidine kinase promoter. This segment contains two Sp1 binding sites. Site directed mutagenesis and transfection studies indicated that both Sp1 sites are functional and both are required for FGFR1 promoter activity. Furthermore, Sp1 binding to the two sites was synergistic enhancing FGFR1 promoter activity. The specificity of Sp1 binding to the two distal promoter cis-elements was verified by electromobility shift and transfection assays employing an Sp1 expression construct. Differences in myoblast versus fibroblast-specific protein-DNA complex formation at these sites correlated with high promoter activity in myoblasts and significantly reduced promoter activity in fibroblasts. These studies for the first time establish a molecular mechanism regulating FGFR1 gene expression during myoblast proliferation.