The immunostimulatory role of Kupffer cells in various inflammatory liver diseases is still not fully understood. In this study, phenotypic and functional aspects of Kupffer cells from healthy C57BL/6 mice were analyzed and compared with those of splenic and peritoneal macrophages to generate a blueprint of the cells under steady-state conditions. In the mouse liver, only one population of Kupffer cells was identified as F4/80(high)CD11b(low) cells. We observed that freshy isolated Kupffer cells are endocytic and show a relatively high basal ROS content. Interestingly, despite expression of TLR mRNA on Kupffer cells, ligation of TLR4, TLR7/8, and TLR9 resulted in a weak induction of IL-10, low or undetectable levels of IL-12p40 and TNF, and up-regulation of CD40 on the surface. Kupffer cells and splenic macrophages show functional similarities, in comparison with peritoneal macrophages, as reflected by comparable levels of TLR4, TLR7/8, and TLR9 mRNA and low or undetectable levels of TNF and IL-12p40 produced upon TLR ligation. The unique, functional characteristics of Kupffer cells, demonstrated in this study, suggest that Kupffer cells under steady-state conditions are specialized as phagocytes to clear and degrade particulates and only play a limited immunoregulatory role via the release of soluble mediators. J. Leukoc. Biol. 92: 723-733; 2012.