The experiments presented in this report were designed to specifically examine the role of CD4-major histocompatibility complex (MHC) class II interactions during T cell development in vivo. We have generated transgenic mice expressing class II molecules that cannot interact with CD4 but that are otherwise competent to present peptides to the T cell receptor. MHC class II expression was reconstituted in Abeta gene knock-out mice by injection of a transgenic construct encoding either the wild-type I-Abetab protein or a construct encoding a mutation designed to specifically disrupt binding to the CD4 molecule. We demonstrate that the mutation, EA137 and VA142 in the beta2 domain of I-Ab, is sufficient to disrupt CD4-MHC class II interactions in vivo. Furthermore, we show that this interaction is critical for the efficient selection of a complete repertoire of mature CD4(+) T helper cells as evidenced by drastically reduced numbers of conventional CD4(+) T cells in animals expressing the EA137/VA142 mutant I-Ab and by the failure to positively select the transgenic AND T cell receptor on the mutated I-Ab. These results underscore the importance of the CD4-class II interaction in the development of mature peripheral CD4(+) T cells.