Major histocompatibility complex (MHC) class I and class II molecules have been shown to present peptides of different origin to alpha beta T cells. Most peptides presented by class I molecules are derived from endogenously synthesized proteins, whereas most peptides presented by class II molecules are from exogenous sources. This functional dichotomy can largely be achieved by the preferential intracellular association of the invariant chain (Ii) with MHC class II molecules, which may inhibit binding of endogenous peptides to class II molecules and direct them to endocytic compartments where extracellularly derived peptides can be sampled. Here, we show that Ii also can associate with a subset of MHC class I molecules and direct them to endocytic compartments. Ii was coprecipitated with class I molecules after lysis of human lymphocytes in mild detergent such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid or digitonin, and the association was more clearly visualized by the use of dithiobis[succinimidyl-propionate], a homobifunctional chemical cross-linker. The class I.Ii complex was reconstituted in Ii negative cells by transfection of corresponding cDNA clones and was found to be transported through the Golgi to acidic endocytic compartments. These observations may explain how some exogenous antigens can be presented by MHC class I molecules and how MHC class II molecules can bind self peptides derived from MHC class I molecules in endocytic compartments.