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The contribution of Gal alpha 1,3Gal (alpha-Gal) to cell-mediated organ xenograft rejection is controversial. We have used recombinant lentiviruses encoding a porcine alpha 1,3 galactosyltransferase (alpha 1,3GalT) to obtain alpha-Gal-expressing primary human aortic endothelial cells (HAEC) at a frequency of 70-90%. These cells were compared to non-transduced and mock-transduced HAEC with regard to their susceptibility to human NK cell-mediated lysis, ability to stimulate IFN-gamma production by NK cells, and support of NK cell adhesion under static and dynamic conditions. Using green fluorescent protein (GFP) as a reporter gene, it was shown that the frequency of green fluorescent HAEC increased until day 5 post-transduction, and at a multiplicity of infection of 2.5, it reached 98%. Lentiviral transduction did not result in activation of HAEC, and transduced HAEC responded as expected to TNF-alpha and IFN-gamma stimulation. No differences were detected between non-alpha-Gal- and alpha-Gal-expressing HAEC in terms of their susceptibility to NK cell-mediated lysis, ability to stimulate IFN-gamma production by NK cells, or ability to support NK cell adhesion under static and dynamic conditions. In conclusion, these data argue against an important role for the alpha-Gal epitope in the direct interaction between endothelium and NK cells and prove that recombinant lentiviruses are efficient gene carriers for primary human endothelial cells.