The invariant chain (li) binds nascent major histocompatibility complex (MHC) class II molecules, blocking peptide binding until the complex dissociates in the endosomes. This may serve to differentiate the MHC class I and II antigen presentation pathways and enable class II molecules to efficiently bind peptides in the endosomes. This hypothesis was addressed by probing spleen cells from a combination of knock-out and transgenic mice with a large panel of T cell hybridomas. The li molecule blocked the presentation of a range of endogenously synthesized epitopes, but some epitopes actually required li. Thus, the influence of li on presentation does not follow simple rules. In addition, mice expressing li were not tolerant to epitopes unmasked in its absence, a finding with possible implications for autoimmunity.