We developed an expression system that aimed to increase the proportion of high producers in a transfected cell population in order to reduce the effort in clone screening. The principle is based on the impairment of the selection marker. Twelve single-point mutations in more or less conserved domains of the resistance marker gene neomycin-phosphotransferase (NPT) resulted in different degrees of reduced enzyme activity, depending on the amino acid conservation and the kind of amino acid exchange. In all transfected, mutant-NPT bearing CHO-DG44 cell pools surviving the selection with G418, the ratio of high-producing cells to total cell number was higher than in pools selected with wildtype-NPT. Furthermore, these pools showed, in comparison to wildtype-NPT selected pools, not only higher NPT-RNA levels but also increased specific productivities and higher titers of a coexpressed biopharmaceutically relevant product. Elevated productivity could be ascribed to higher gene copy numbers, integration into chromatin regions with higher transcriptional activity, or a combination of both effects. Thus, the use of NPT-mutants as selection markers is suitable for the enrichment of high producers in a transfected CHO-DG44 cell population, since cell survival is achieved only if the enzymatic impairment of the cointegrated resistance marker is compensated by a higher expression level. (C) 2005 Wiley Periodicals, Inc.