Introduction: Endomyocardial biopsy is the standard means of establishing cardiac allograft rejection diagnosis. The efficacy of this procedure in xenotransplantation has not been determined. In this study we compare the histology of right ventricular endomyocardial biopsy specimens with the corresponding full cross sections of explanted right ventricle (RV). We also compare RV with the related left ventricle (LV) cross sections. Methods: Heterotopic CD46 pig-to-baboon cardiac xenotransplants (n = 64) were studied. RV endomyocardial biopsy specimens were taken at cardiac explant by using a standard bioptome (n = 24) or by sharp dissection (n = 40). Hematoxylin and eosin stained sections of RV and LV cross-section and RV endomyocardial biopsy specimens were compared in a blinded fashion. Characteristics of delayed xenograft rejection and a global assessment of ischemia were scored from 0 to 4 according to the percentage of myocardium involved (0, 0%; 1, 1%-25%; 2, 26%-50%; 3, 51%-75%; and 4, 76%-100%). Results: Median graft survival was 30 days (range, 3-137 days). Linear regression analysis of histology scores demonstrated that specimens from both bioptome and sharp dissection equally represented the histology of the RV cross section. Global ischemic injury was strongly correlated between RV and RV endomyocardial biopsy (R2 = 0.84) and between RV and LV cross sections (R2 = 0.84). Individual characteristics of delayed xenograft rejection showed no significant variation between RV and RV endomyocardial biopsy or between RV and LV (p < 0.05). Conclusions: These results indicate that delayed xenograft rejection is a widespread process involving both right and left ventricles similarly. This study shows that histologic assessment of RV endomyocardial biopsy specimens is an effective method for the monitoring of delayed xenograft rejection after cardiac xenotransplantation. © 2007 International Society for Heart and Lung Transplantation.