The analysis of T cell receptor alpha (TCR alpha) chains in mice transgenic for a TCR beta chain has allowed us to demonstrate a central role for self-peptides in the positive intrathymic selection of major histocompatibility complex (MHC) class II-restricted T cells. Analysis of specific V alpha-J alpha joins in mature CD4+ TCRhigh thymocytes and in peripheral CD4+ T cells revealed a limitation in amino-acid sequences. By analysis of immature thymocytes, we could show that this limited repertoire was selected from a more diverse repertoire. By analysis of the same beta chain-transgenic mice bred to H-2Ma-deficient mice that express one or a very limited number of peptides, we could demonstrate that the V alpha-J alpha join repertoire was now altered and much more limited. Together, these data provide molecular and genetic evidence that the intrathymic positive selection of the TCR repertoire is critically affected by self-peptides presented by MHC class II molecules, most likely on thymic cortical epithelial cells.