The successful application of cancer gene therapy has been hampered by the low efficiency of in vivo gene delivery by currently used replication-defective vectors. Accordingly, considerable efforts are now being directed toward development and use of vectors capable of replicating in cancer cells. However, for replicating retroviruses, insertion of additional reading frames into the viral genome often resulted in the generation of unstable viruses. Here, we report a novel concept for the generation of replication-competent murine leukemia virus (MLV) vectors capable of mediating the secretion of soluble therapeutic proteins from infected cells. As a proof of principle, we inserted transgene regions encoding either a single-chain variable region fragment (scFv), here, the laminin-specific L36-scFv, or the T-cell-specific 7A5-scFv, or the cytokine GM-CSF into the MLV envelope (env) gene after +1 codon of the envelope (Env) protein, followed by a sequence specifying a furin protease cleavage site. The resulting viruses, termed L36-furin-A, 7A5-furin-A and GMCSF-furin-Mo, respectively, infected a variety of human cell lines, including HMEC-1 (endothelial), A301 (lymphoid), MDA-MB231 and MDA-MB468 (breast cancer) and HT1080 (fibrosarcoma) cells. Western blot analysis of conditioned culture medium from HT1080 cells infected by replicating L36-furin A, as an example, revealed that more than 90% of the Env fusion protein molecules were indeed intracellularly cleaved. After 5 days of infection, up to 3-4 mug/ml of soluble L36-scFv accumulated in the supernatant of HT1080 cells. The eukaryotically produced L36-scFv and 7A5-scFv were able to recognize their native antigens with high avidity, as assessed by ELISA and flow cytometry. Furthermore, the replicating viruses were genetically stable for more than 12 cell passages. In conclusion, a new generation of replication-competent retroviral vectors capable of mediating long-term and efficient secretion of therapeutic proteins suitable for cancer therapy was generated.