Efficient selection of gene-modified cells is required for a number of potential gene therapy applications, as well as molecular biology studies. Ideally, a clinical selection regimen would combine high selection speed, efficiency and efficacy, in addition to clinical grade selection techniques and low immunogenicity. To our knowledge, a selection marker satisfying all these features is so far not available. Ouabain is a clinically used cardiac glycoside and selective Na(+)/K(+)-ATPase inhibitor. On the basis of the high sensitivity of human Na(+)/K(+)-ATPase proteins to ouabain, and rapid killing of cells upon exposure, we have screened the ubiquitously expressed Na(+)/K(+)-ATPase alpha1 subunit for mutations that could greatly increase its resistance to ouabain. Two amino-acid substitutions, Q118R and N129D were sufficient to confer a two log greater resistance to ouabain in HeLa, Jurkat, U2OS cells and in primary cells. Furthermore, following transduction of primary lymphocytes with the alpha1(Q118R/N129D) gene, >99% pure populations of gene-modified cells were achieved with a recovery rate of >80% after 48 h of exposure to ouabain. These results identify the human alpha1(Q118R/N129D) (OuaSelect) as a promising selection marker gene for safe, rapid and cost-effective selection in clinical gene therapy and molecular biology research.