Mastitis affects the concentrations of potassium and sodium in milk. Since sodium-potassium adenosine triphosphatase (Na(+), K(+)-ATPase) is critical for maintaining the homeostasis of these two ions, and is involved in cell apoptosis and pathogenesis, we presumed that polymorphism of the ATP1A1 gene, which encodes the bovine Na(+), K(+)-ATPase α1 subunit could be associated with mastitis. The ATP1A1 gene was analyzed in 320 Holstein cows using PCR low ionic strength single-strand conformation polymorphism (PCR-LIS-SSCP) and DNA sequencing methods. A C/A SNP was identified at nucleotide position -15,739 in exon 17 of the ATP1A1 gene, but it did not induce any change in amino acids. We examined a possible association of polymorphism of the ATP1A1 gene with somatic cell score and 305-day milk yields. Individuals with genotype CC in ATP1A1 had significantly lower somatic cell scores and 305-day milk yields than those with genotype CA. We also examined changes in Na(+), K(+)-ATPase activity of red cell membranes. The Na(+), K(+)-ATPase activity was significantly higher in dairy cows with genotype CC compared to the other two genotypes, and the Na(+), K(+)-ATPase activity of the resistant group was significantly higher than that of the susceptible group in dairy cows. We conclude that this polymorphism has potential as a marker for mastitis resistance in dairy cattle.