Ag receptor gene loci contain many V gene segments, each of which is recombined and expressed at a different frequency and is subject to allelic exclusion. To probe the parameters that mediate the different levels of regulation of V gene rearrangement, a Vβ gene segment together with 3.6-kb 5′ and 0.7-kb 3′ flanking sequences was inserted 6.8 kb upstream of the Dβ1 gene segment in the murine TCRβ locus. Despite its proximity to the Dβ gene segments and the Eβ enhancer, the inserted Vβ segment underwent VDJ recombination at the same frequency as the natural copy located 470 kb upstream. However, the inserted Vβ segment was no longer under allelic exclusion control as it recombined at a similar frequency in the presence of a TCRβ transgene. These results suggest that while the inserted fragment contains the necessary cis-regulatory elements for determining the frequency of Vβ rearrangement, additional cis-regulatory elements are required for mediating Vβ allelic exclusion. Interestingly, most of the inserted Vβ rearrangements were not transcribed and expressed in the presence of a TCRβ transgene, suggesting that TCRβ allelic exclusion can also be achieved by blocking the transcription of the rearranged gene segments. These findings provide strong evidence for distinct control of the frequency and allelic exclusion of Vβ gene rearrangement.