Patients after polytrauma, burns, or septic shock frequently develop a life-threatening immunodeficiency. This state is associated with specific functional alterations of monocytic cells. We previously proposed endotoxin tolerance, the monocyte state after acute response to lipopolysaccharide, as a respective model system. One major feature in both the clinical situation and the in vitro model is the dramatic down-regulation of monocyte major histocompatibility complex (MHC) class II surface expression, which is associated with impaired antigen presentation capacity. This study focused on the mechanisms behind reduced MHC class II expression in endotoxin tolerance. Endotoxin priming provoked a decrease of monocyte intracellular MHC class II. It also led to a reduced expression of the chaperonic invariant chain and to an inhibited synthesis of the major lysosomal enzyme for final cleavage of the invariant chain going along with a relative accumulation of p10. The expression of HLA-DM necessary for loading MHC class II with antigenic peptide was also decreased. Additionally, reduced export of MHC class II alphabeta complexes to the cell surface was observed. The down-regulation of HLA-DR, invariant chain, and HLA-DM was regulated at the mRNA level and may be the consequence of reduced class II transactivator expression observed in this study. The simultaneous interference at different regulatory levels may explain the uniquely strong and long lasting MHC class II down-modulating effect of endotoxin priming compared with transforming growth factor-beta and interleukin-10. These results not only contribute to a better understanding of experimental endotoxin tolerance but may also give rise to new therapeutics for temporary immunodeficiency and, conversely, for MHC class II-dependent diseases such as autoimmunity and transplant rejection.