The appearance of peptide-loaded major histocompatibility complex (MHC) class II molecules at the cell surface depends critically on the invariant chain (Ii). We have studied the influence of Ii on the positive selection of CD4+ T cells, mediated by class II molecules expressed on thymic stromal cells. Invariant chain-deficient mice (Ii°) were crossed with different T cell receptor (TcR) transgenic strains and the emergence of mature CD4 single-positive thymocytes measured in Ii°/TcR transgenic offspring. Positive selection was nearly absent in Ii°/2B4 mice, which display receptors specific for a moth cytochrome c (MCC) peptide in the context of Ek. In addition, no T cell response was elicited when nontransgenic Ii° animals were injected with this peptide, even though antigenpresenting cells (APC) from such mice were perfectly capable of presenting it, suggesting that selection of the entire anti-MCC 88-103 repertoire depends on Ii. Positive selection also appeared strongly reduced in another line of Ii°/TcR transgenic mice (Ii°/BDC2.5). However, in sharp contrast, a third line (Ii°/3A9) exhibited almost normal positive selection of thymocytes displaying the transgene-encoded receptor. These thymocytes were exported to the periphery; peripheral T cells could respond normally to the appropriate peptide in vitro. The most likely interpretation of these findings is that selection of most CD4+ T cells depends on MHC class II complexes loaded with peptide in an Ii-dependent pathway, but some can be selected on class II complexes that are either loaded along an alternative, Ii-independent, route or are empty. This is consistent with the involvement of peptide in positive selection of CD4+ T cells, for which there exists little prior evidence.