We have determined the sequence of the 5′-flanking region and first three exons of the human Na,K-ATPase α1 gene, ATP1A1. Primer extension and S1 nuclease protection analyses of RNA from human kidney, brain, and skeletal muscle indicate that transcription initiates 273 nucleotides upstream of the translation start site. The promoter region contains a potential TATA box at position −27 relative to the transcription initiation site; however, no CCAAT sequence is observed. The 5′-untranslated and 5′-flanking regions are G + C rich. Five sequence elements exhibiting similarity to binding sites for the transcription factor Sp 1 are located within the 5′-flanking region. This region also contains potential binding sites for the transcription factors AP-1, AP-2, AP-3, and NF-1, as well as a site which exhibits perfect identity to an 8-bp sequence element important for calcium induction. A comparison of the 5′-flanking region of the α1 and α2 genes reveals differences in potential transcription factor and hormone receptor binding sites which may be important in mediating the tissue- and developmental stage-specific expression of these genes. We have also identified an intragenic DNA probe which detects a restriction fragment length polymorphism at the α1 locus. This marker should facilitate genetic linkage studies designed to evaluate the role of the sodium pump in human disease.