The influence of individual peptides in thymic selection was examined in H2-M- mice, in which positive selection is directed to a single peptide, class II-associated invariant chain peptide (CLIP) bound to H2-A(b). Two sensitive in vivo approaches showed that 70%-80% of CD4+ T cells undergoing positive selection to CLIP+H2-A(b) have self-reactivity to the various peptides expressed on wild-type H2-M+ antigen-presenting cells. When these self-reactive T cells were depleted, the residual CD4+ cells displayed a polyclonal repertoire in terms of alloreactivity, responses to foreign protein antigens, and Vbeta usage. Nevertheless, studies with two T cell receptor transgenic lines suggested that the repertoire of CD4+ cells induced by CLIP was less diverse than the repertoire of CD4+ cells in normal mice. Generation of a fully diverse T cell repertoire thus requires positive selection against multiple peptides.