V(D)J recombination is targeted by short recombination signal (RS) sequences that are relatively conserved but exhibit natural sequence variations. To evaluate the potential of RS sequence variations to determine the primary and peripheral TCRbeta repertoire, we generated mice containing specific replacement of the endogenous Vbeta14 RS with the 3'Dbeta1 RS (Vbeta14/3'DbetaRS). These mice exhibited a dramatic increase in Vbeta14(+) thymocyte numbers at the expense of thymocytes expressing other Vbetas. In addition, the percentage of peripheral Vbeta14(+) alphabeta T lymphocytes was similarly increased. Strikingly, this altered Vbeta repertoire resulted predominantly from a higher relative level of primary Vbeta14/3'DbetaRS rearrangement to DbetaJbeta complexes, despite the ability of the 3'Dbeta1 RS to break B12/23 restriction and allow direct rearrangement of Vbeta14/3'DbetaRS to Jbeta segments.