To characterize the ligand binding properties of a naive T cell repertoire capable of responding to a foreign antigen, we analyzed T cell populations from T cell receptor (TCR) beta transgenic mice using a novel, single cell peptide/major histocompatibility complex (MHC) tetramer dissociation assay. The largely CD4+CD8(-/low) antigen-specific thymocyte repertoire exhibited a broad, bimodal distribution of tetramer binding half-lives (t(1/2)s), with a significant underrepresentation in the intermediate half-life range in which the majority of the peripheral repertoire lies. Thus, cells with the potential to bind foreign antigen with the lowest and highest stability are likely to be selectively removed from the repertoire prior to their establishment in the periphery. These studies provide direct evidence that thymic selection biases the naive peripheral T cell repertoire toward TCR-ligand interactions that fall within a moderate half-life "window."