We identified cis elements in the 5′-flanking region of rat Na,K-ATPase α2 subunit gene (Atp1 a2) using transient transfection assays in L6 rat skeletal muscle myoblast cells. By 5′-deletion mutation analysis, the region between nucleotide positions –175 and –108 was identified as a positive regulatory region. In the region, the distal E box (nucleotides –144 to –139) acts as a negative regulatory element, and the Sp1 consensus sequence (nucleotides –123 to –118) and the GGGAGG sequence (nucleotides –114 to –109) act as positive regulatory elements. Gel-retardation analysis revealed that binding factors are an E-box-binding protein and Sp1. DNase I foot-printing and methylation-interference analyses revealed that Sp1 binds to the region from nucleotides –122 to –101 and the E-box-binding protein to the region from nucleotides –144 to –136. T4 DNA polymerase footprinting revealed that there are three Sp1-binding sites in the region and that Sp1 binds to one of the three sites in a mutually exclusive manner. The mechanism by which Sp1 activates the Atp1a2 promoter is discussed.