Cell-mediated immune mechanisms underlying discordant xenograft rejection are poorly characterized. In our study, using a human to rat xenogeneic ex vivo model, we show that a fraction of human lymphocytes, when perfused through the coronary system of a rat heart, rapidly and specifically adheres to the vascular endothelium and infiltrates the myocardium. Lymphocyte phenotypic analysis before and after perfusion, as well as the use of purified cell subpopulations, demonstrate preferential adhesion of CD3- CD16+ NK cells. NK cell adhesion occurs via xenoreactive antibody-dependent and -independent pathways, because the selective removal of human IgG from the perfusion buffer markedly reduces but does not completely abrogate NK cell sequestration. However, T lymphocytes are retained in the xenoorgan via an antibody-independent pathway, as assessed by the lack of influence of IgG removal. Leukocyte integrins appear to play a crucial role in mediating adhesion of both lymphocyte subsets, because the pretreatment of lymphocytes with anti-CD11a, anti-CD11b, and anti-CD18 antibodies markedly reduces their retention into the xenogeneic organ. Retained human lymphocytes mediate rapid and direct damage of the xenoorgan, as demonstrated by histologic and functional alterations of the endothelium, impaired vascular resistance and in vitro lysis of rat endothelial cells by human NK cells. Taken together, these findings suggest a role for cell-mediated mechanisms in the rapid recognition and rejection of vascularized xenografts.