Hyperacute rejection (HAR) currently precludes the use of discordant organs for human transplantation. In order to comprehensively evaluate HAR in a clinically applicable species combination, we have developed an ex vivo perfusion model utilizing a neonatal extracorporeal membrane oxygenator circuit; this model allows for functional and sequential biopsy studies of working piglet hearts sustained by human, single donor AB+ type blood. A detailed description of the methods employed is included. Hearts perfused by allogeneic pig blood sustained normal function throughout the study period, while those perfused with human blood lost organized ventricular contraction in 25-34 min with markedly attenuated function. Compared with biopsies from piglet hearts perfused with allogeneic blood and biopsies taken prior to human blood perfusion (t = 0), biopsies of hearts perfused with human blood at t = 15 and 30 min demonstrated significant inflammatory changes involving vessels (endothelial and myointimal swelling and reaction) as well as myocardium (injury and necrosis). By immunohistology, significant vascular deposition of IgM, IgG, fibrinogen, C3, and C1q was seen, along with infiltrates of human leukocytes consisting predominantly of neutrophils, macrophages, and T cells, with occasional B cells and NK cells. Sequential studies of circulating blood demonstrated the progressive consumption of human leukocytes and human anti-porcine antibodies, but no decrease in complement activity as measured by CH50. These findings indicate that the rapid loss of function seen in human anti-porcine cardiac HAR is associated with deposition of IgM and IgG xenoreactive antibody and early complement components and that extensive infiltration by inflammatory cells occurs within 15-30 min. This model provides a useful system for the study of human anti-porcine HAR.