The objective of this study was to investigate the efficacy of combination gene therapy with multiple angiogenic growth factor cDNAs to enhance survival of ischemic skin flaps in a rat model. Sixty Sprague-Dawley rats were divided into six groups. Varying combinations of VEGF165, PDGF-B, and bFGF-plasmids were injected to prefabricate the flaps. Random skin flaps were raised on the dorsal aspect of rats following prefabrication with growth factor cDNAs. Flap viability was determined by measurement of percentage area of survival. The efficacy of gene therapy was evaluated by flap survival and neovascularization of representative histologic sections stained immunohistologically. The VEGF165 plus bFGF cDNAs enhanced the viability of the flap and neovascularization most effectively; the flap survival area was 64.3 +/- 8.7% after transfer of these two growth factor genes. Addition of PDGF-B cDNA is deleterious to the effects of combined VEGF165 and bFGF, leading to a significant decrease in flap viability (44.9 +/- 2.7%). Viability of the flaps with combined VEGF165 and bFGF cDNA transfer was significantly greater than that of the flaps with VEGF165 transfer alone (57.6 +/- 5.2%) or sham plasmid control (52.3 +/- 5.0%). Combined transfer of VEGF165 and bFGF cDNA is the most effective combination of multiple growth factor genes to improve flap viability in this model. Simultaneous transfer of three growth factor genes (VEGF165, PDGF-B, and bFGF) is deleterious to flap survival, at least for the ratio of lipofectin:transgene employed.