It is generally accepted that donor myeloid dendritic cells (MDC) are the main instigators of acute rejection after organ transplantation. The aim of the present study was to characterize MDC in human donor livers using liver grafts and perfusates as a source. Perfusates were collected during ex vivo vascular perfusion of liver grafts pretransplantation. MDC, visualized in wedge biopsies by immunohistochemistry with anti-BDCA-1 monoclonal antibody (mAb), were predominantly observed in the portal fields. Liver MDC, isolated from liver wedge biopsies, had an immature phenotype with a low expression of CD80 and CD83. Perfusates were collected from 20 grafts; perfusate mononuclear cells (MNC) contained 1.5% (range, 0.3-6.6%) MDC with a viability of 97 ± 2%. Perfusates were a rich source of hepatic MDC since 0.9 × 106 (range, 0.11-4.5 × 106) MDC detached from donor livers during vascular perfusion pretransplantation. Perfusate MDC were used to further characterize hepatic MDC. Perfusate MDC expressed less DC-LAMP (P = 0.000), CD80 (P = 0.000), CD86 (P = 0.003), and CCR7 (P = 0.014) than mature hepatic lymph node (LN) MDC, and similar CD86 (P = 0.140) and CCR7 (P = 0.262) as and more DC-LAMP (P = 0.007) and CD80 (P = 0.002) than immature blood MDC. Perfusate MDC differed from blood MDC in producing significantly higher amounts of interleukin (IL)-10 in response to lipopolysaccharide (LPS), and in being able to stimulate allogeneic T-cell proliferation. In conclusion, human donor livers contain exclusively immature MDC that detach in high numbers from the liver graft during pretransplantation perfusion. These viable MDC have the capacity to stimulate allogeneic T-cells, and thus may represent a major player in the induction of acute rejection. Liver Transpl 12: 384–393, 2006. © 2006 AASLD.