The genetic transfer of drug resistance to hematopoietic cells is an attractive approach to overcoming myelosuppression caused by high-dose chemotherapy. Because cyclophosphamide (CTX) and methotrexate (MTX) are commonly used non-cross-resistant drugs, generation of dual drug resistance in hematopoietic cells that allows dose intensification may increase anti-tumor effects and circumvent the emergence of drug-resistant tumors. We constructed a retroviral vector containing both a human cytosolic ALDH-1 cDNA and a human doubly mutated DHFR cDNA (Phe22/Ser31; termed F/S in the description of constructs) to generate increased resistance to both CTX and MTX. Infection of NIH3T3 cells resulted in increased resistance to both 4-hydroperoxycyclophosphamide (4HC) (1.9 +/- 0.1-fold) and MTX (73 +/- 2.8-fold). Transduced human CD34(+) enriched hematopoietic progenitor cells were also resistant to both 4HC and MTX by CFU-GM readout. Lethally irradiated mice transplanted with SFG-ALDH-IRES-F/S or mock-transduced bone marrow cells were treated with high-dose pulse CTX or high-dose CTX/MTX. Animals receiving marrow not transduced with ALDH-1 or mutated DHFR cDNA died from CTX or CTX/MTX toxicity, whereas mice transduced with ALDH-1 and mutated DHFR cDNA-containing marrow were able to tolerate the same doses of CTX or CTX/MTX treatment posttransplant. These data taken together indicate that ALDH-1 overexpression and mutant DHFR increased both 4HC and MTX resistance in vitro and in the in vivo mouse model. This construct may be useful for protecting patients from high-dose CTX- and MTX-induced myelosuppression.