To develop a gene therapy protocol suitable for the treatment of a benign disease such as Gaucher disease, we have developed two bicistronic vectors that allow transduced cells to be selected for with methotrexate (MTX). The two vectors differ in the presence or absence of a mutant polyoma enhancer (ΔMo+PyF101) replacing the wild-type retroviral enhancer in the LTR. Infection of human TF-1 and K562 cells, Gaucher type II fibroblasts and murine hemopoietic bone marrow cells conferred MTX resistance and glucocerebrosidase (GC) expression. Upon increasing MTX concentrations, the number of proviral copies and GC activity increased, demonstrating in vitro selection of retrovirus-transduced cells. At high MTX selection pressure, up to 140 μM for infected Gaucher type II fibro- blasts, no endogenous wild-type DHFR amplification could be detected, indicating that both retroviral constructs provide sufficient DHFR protein levels. Upon transduction, murine bone marrow cells were protected against otherwise lethal MTX concentrations (range 1–5 μM MTX). Flow cytometry specific for human GC (hGC) demonstrated that in vitro selection resulted in increased percentages of hGC-positive murine cells. In conclusion, the generated bicistronic vectors are ideally suited to investigate whether an in vivo selection approach for retrovirus-transduced cells is feasible. Such a strategy might abolish the need for a high initial transduction efficiency and might result in a gene therapy protocol devoid of the undesirable need for marrow ablative treatment of the recipient.