Although it is well established that antigen dose plays an important role in determining the quality of T cells induced in vitro, it has not well been determined whether antigen dose also affects T cell repertoires induced in vivo. This study demonstrates that variation of antigen doses in vivo as well as in vitro induce structurally and functionally different T cell repertoires. CTLs generated in vitro with a low antigen dose showed much higher T cell responsiveness than CTLs generated with a high antigen dose, and the two CTL populations employed different TCR Vbeta chains. This is most likely due to repertoire selection based on TCR affinity. The secondary in vivo responses with a high or low dose of antigen following the primary response raised with the same dose resulted in a reversed dominance pattern of two particular TCR Vbeta phenotypes. TCR affinity of these two T cell populations appeared different, suggesting avidity selection based on antigen availability. Indeed, they required a distinct level of antigen for maximal cytolytic function, implying a different functional avidity. These results suggest that antigen-specific T cell repertoire is substantially affected by the antigen dose employed in vivo as well as in vitro.