T-cell receptor (TCR) beta variable region exons are assembled from numerous gene segments in a highly ordered and regulated manner. To elucidate mechanisms and identify cis-acting elements that control Vbeta rearrangement, we generated an endogenous TCR-beta allele with only the Vbeta2, Vbeta4, and Vbeta14 segments. We found that alphabeta T lineage cells containing this Vbeta(2-4-14) allele and a wild-type TCR-beta allele developed normally, but exhibited a significant increase in Vbeta2(+) and Vbeta14(+) cells. To quantify Vbeta rearrangements on the Vbeta(2-4-14) allele, we generated alphabeta T-cell hybridomas and analyzed TCR-beta rearrangements. Despite the deletion of almost all Vbeta segments and 234 kb of Vbeta cluster sequences, the Vbeta(2-4-14) allele exhibited only a slight decrease in Vbeta rearrangement as compared with the wild-type TCR-beta allele. Thus, cis-acting control elements essential for directing Vbeta rearrangement across large chromosomal distances are not located within the Vbeta cluster. We also found a significant increase in the frequency of Vbeta rearrangements involving Vbeta2 and Vbeta14, but not Vbeta4, on the Vbeta(2-4-14) allele. Collectively, our data suggest that Vbeta cluster sequences reduce the frequency of Vbeta2 and Vbeta14 rearrangements by competing with the productive coupling of accessible Vbeta2 and Vbeta14 segments with DJbeta1 complexes.