The major histocompatibility (MHC) class II molecules present peptides to the T cell receptor on CD4+ lymphocytes. The diversity of the epitopes presented depends critically on the processing of the ingested antigens, and the endocytic pathway constitutes a set of increasingly protease-rich/acidic compartments to facilitate the progressive digestion of antigens. Targeting of newly synthesized MHC class II molecules to more acidic endosomal compartments depends on association with the invariant chain (Ii) molecule. Ii is synthesized in excess of class II and is also present in a MHC class II-free form. It is therefore possible that Ii may have other functions in post-endoplasmatic reticulum compartments. In this study we demonstrate the interaction between free Ii and mature MHC class II molecules present at the plasma membrane. Using a photoactivatable green fluorescent protein (PAGFP) fused to MHC class II in combination with an inducible expression system for Ii we show that excessive amounts of Ii causes an increased endocytosis of mature class II, and the redistribution of these to late endosomes and lysosomes. This secondary association will thereby expand the repertoire of peptides encountered by mature MHC class II molecules.