AbstractProgress in FGF‐2 gene therapy has been hampered by the difficulty in achieving therapeutic levels of FGF‐2 secretion. This study tested whether the addition of BMP2/4 hybrid secretion signal to the FGF‐2 gene and mutation of cys‐70 and cys‐88 to serine and asparagine, respectively, would increase the stability and secretion of active FGF‐2 protein in mammalian cells using MLV‐based vectors. Single or double mutations of cys‐70 and cys‐88 to ser‐70 and asp‐88, respectively, markedly increased the amounts of FGF‐2 protein in conditioned media and cell lysates, which may be due to glycosylation, particularly at the mutated asp‐88 residue. Addition of BMP2/4 secretion signal increased FGF‐2 secretion, but also suppressed FGF‐2 biosynthesis. The combination of BMP2/4 secretion signal and double cys‐70 and cys‐88 mutations increased the total amount of secreted FGF‐2 protein >60‐fold. The modifications did not alter its ability to stimulate cell proliferation and Erk1/2 phosphorylation in marrow stromal cells or its ability to bind heparin in vitro, suggesting that the modified FGF‐2 protein was functionally as effective as the unmodified FGF‐2. An ex vivo application of rat skin fibroblasts (RSF) transduced with the modified FGF‐2 vector in a subcutaneous implant model showed that rats with implants containing cells transduced with the modified FGF‐2 vector increased serum FGF‐2 level >15‐fold, increased growth of the implant, and increased vascularization within the implant, compared to rats that received implants containing β‐galactosidase‐ or wild‐type FGF‐2‐transduced control cells. This modified vector may be useful in FGF‐2 gene therapy investigations. J. Cell. Biochem. 100: 1493–1508, 2007. © 2007 Wiley‐Liss, Inc.