Cross‐presentation is an important function of immune competent cells, such as dendritic cells (DCs), macrophages, and an organ‐resident liver cell population, i.e., liver sinusoidal endothelial cells (LSECs). Here, we characterize in direct comparison to DCs the distinct dynamics and kinetics of cross‐presentation employed by LSECs, which promote tolerance induction in CD8 T cells. We found that LSECs were as competent in cross‐presenting circulating soluble antigen ex vivo as DCs at a per‐cell basis. However, antigen uptake in vivo was 100‐fold more pronounced in LSECs, indicating distinct mechanisms of cross‐presentation. In contrast to mannose‐receptor–mediated antigen uptake and routing into stable endosomes dedicated to cross‐presentation in DCs, we observed distinct antigen‐uptake and endosomal routing with high antigen turnover in LSECs that resulted in short‐lived cross‐presentation. Receptor‐mediated endocytosis did not always lead to cross‐presentation, because immune‐complexed antigen taken up by the Fc‐receptor was not cross‐presented by LSECs, indicating that induction of CD8 T cell tolerance by LSECs is impaired in the presence of preexisting immunity. Conclusion: These results provide a mechanistic explanation how organ‐resident LSECs accommodate continuous scavenger function with the capacity to cross‐present circulating antigens using distinct kinetics and dynamics of antigen‐uptake, routing and cross‐presentation compared to DCs. (HEPATOLOGY 2009.)