Expression of Na,K-ATPase activity is up-regulated in cells incubated for extended intervals in the presence of low external K+. Our previous data showed that exposure of cardiac myocytes to low K+ increased the steady-state abundance of Na,K-ATPase β1 subunit mRNA. In the present study we determined that incubation of primary cultures of neonatal rat cardiac myocytes with low K+ augmented Na,K-ATPase β1 gene expression at a transcriptional level and that this effect required extra-cellular Ca2+. The stimulatory effect of low K+ on Na,K-ATPase β1 gene transcription was not dependent on increased contractile activity of cardiac myocytes. Na,K-ATPase β1 5'-flanking region deletion plasmids used in transient transfection analysis demonstrated that the region between nucleotides -62 to -42 of the β1 promoter contained a low K+ response element. Site-directed mutagenesis of a potential GC box core motif GCG in the -58/-56 region of the β1 promoter decreased basal and low K+-mediated transcription. Mutation of the core sequence of a putative GC box element located between nucleotides -101 and -99 further decreased the low K+ effect on β1 gene transcription. Electrophoretic mobility shift assays using oligonucleotides spanning the proximal and distal GC box elements of the β1 promoter showed enhanced binding of two complexes in response to low K+. The inclusion of a consensus GC box sequence as a competitor in gel shift analysis reduced factor binding to the low K+ response elements. Antibodies to transcription factors Sp1 and Sp3 interacted with components of both DNA-binding complexes and binding of nuclear factors was abolished in gel shift studies using GC box mutants. Together these data indicate that enhanced binding of Sp1 and Sp3 to two GC box elements in the rat Na,K-ATPase β1 subunit gene promoter mediates β1 gene transcription up-regulation in neonatal rat cardiac myocytes exposed to low external K+.