CD4⁺ T cells are positively selected in the thymus on peptides presented in the context of major histocompatibility complex class II molecules expressed on cortical thymic epithelial cells. Molecules regulating this peptide presentation play a role in determining the outcome of positive selection. Cathepsin L mediates invariant chain processing in cortical thymic epithelial cells, and animals of the I-A^b haplotype deficient in this enzyme exhibit impaired CD4⁺ T cell selection. To determine whether the selection defect is due solely to the block in invariant chain cleavage we analyzed cathepsin L–deficient mice expressing the I-A^q haplotype which has little dependence upon invariant chain processing for peptide presentation. Our data indicate the cathepsin L defect in CD4⁺ T cell selection is haplotype independent, and thus imply it is independent of invariant chain degradation. This was confirmed by analysis of I-A^b mice deficient in both cathepsin L and invariant chain. We show that the defect in positive selection in the cathepsin L^−/− thymus is specific for CD4⁺ T cells that can be selected in a wild-type and provide evidence that the repertoire of T cells selected differs from that in wild-type mice, suggesting cortical thymic epithelial cells in cathepsin L knockout mice express an altered peptide repertoire. Thus, we propose a novel role for cathepsin L in regulating positive selection by generating the major histocompatibility complex class II bound peptide ligands presented by cortical thymic epithelial cells.