Objective: The transfer of recombinant genes to donor organs may allow for novel therapeutic approaches to the challenges of acute and chronic rejection. Adenoviral vectors are capable of efficient gene transfer, but use of these vectors during donor organ preservation may be less efficient due to the low temperature. This study was designed to examine the effect of temperature on the efficiency of adenovirus-mediated gene transfer. Methods: Gene transfer to human endothelial cells, porcine vascular smooth muscle cells and cultured rat thoracic aortas was examined. Incubation with an adenoviral vector encoding for E. coli β-galactosidase was performed for I h at three different temperatures: 4°C, 10°C and 37°C. Transgene expression was assessed by histochemical staining for β-galactosidase in transduced cells and by evaluation of β-galactosidase activity in organ cultures. Results: Both in human endothelial cells and vascular smooth muscle cells the percentage of positively staining cells following transduction at 37°C was significantly greater than at 4°C and at 10°C (30.55 ± 7.26% vs. 14.29 ± 3.79% and 12.43 ± 2.47%, respectively for endothelial cells, P < 0.01 vs. 4°C and 10°C; and 28.25 ± 4.52% vs. 17.91 ± 3.76% and 16.63 ± 3.92%, respectively for smooth muscle cells, P < 0.05 vs. 4°C, P < 0.01 vs. 10°C). β-galactosidase activity was significantly greater in aortas transduced at 37°C than in vessels transduced at 4°C and 10°C (289 700 ± 113 300 vs. 149 600 °54 390 and 108 800 ± 23 140 relative chemiluminesce units/mg of total protein, respectively; P < 0.05 vs. 4°C, P < 0.001 vs. 10°C). Conclusions: The present study demonstrates that the efficiency of adenovirus-mediated gene transfer is significantly reduced at lower temperatures. The need for cold preservation of donor organs may render efficient adenovirus-mediated gent transfer more difficult in the transplantation setting.