Background: Gene transfer to a donor cornea ex vivo can modulate corneal graft failure in experimental animal models. We compared a lentiviral vector (LV) carrying the transgene ovine interleukin 10 (IL10) with a comparable adenoviral vector (Ad) for its ability to transduce ovine and human corneas and to modulate ovine corneal allograft survival. Methods: The LV carrying the ovine IL10 gene was used to transduce ovine and human corneas in vitro. LV-mediated gene expression in corneal endothelium was assessed by real-time quantitative reverse-transcriptase polymerase chain reaction, at varying doses and duration of transduction. The effect of ex vivo transduction of the donor cornea with LV-SV40-IL10 was assessed following orthotopic corneal transplantation in outbred sheep. Results: Expression of IL10 mRNA in Ad-CMV-IL10-transduced ovine corneas was 103-fold higher than in LV-SV40-IL10-transduced corneas (P < 0.0001), and 107-fold higher than in non-transduced controls. IL10 was secreted rapidly from Ad-CMV-IL10-transduced, organ-cultured corneas, peaking at 13-15 days. IL10 secreted from LV-SV40-IL10-transduced corneas increased 20-fold compared with controls, but had not reached a plateau at 15 days. Gene expression driven by LV-SV40-IL10 varied with vector dose and transduction time, but was less than with Ad-CMV-IL10 at both mRNA and protein levels. Gene expression driven by LV-SV40-IL10 was faster in the human cornea than the ovine cornea. Corneal allograft survival was prolonged by a median of 7 days in the LV-SV40-IL10-treated recipients, compared with the control group (P = 0.026). Conclusion: Although lentiviral vectors show some promise for corneal gene therapy, they are less efficient than adenoviral vectors.