The gene coding for a T-cell receptor (TCR) alpha chain is assembled from variable (ADV) and joining (AJ) genes located on Chromosome 14. Each of the 90 ADV genes can rearrange with any one of the 61 AJ genes. We have previously demonstrated that ADV and AJ gene segment use evolves with time, with a progressive opening of ADV and AJ regions of the locus. To define the rules governing the use of AJ genes by ADV genes belonging to one family, we carried out a detailed analysis of 268 combinations of ADV2 BALB/c transcripts. We found that the different ADV2 members use different sets of AJ genes depending on their location within the ADV locus: ADV2S7 (the most AJ proximal ADV2 member) rearranges mainly with the AJ genes located close to the TEA element, whereas 50% of the sequences for ADV2S8, which is distal to the AJ locus, use the most distal AJ genes. ADV2S5, an ADV2 member located in the middle of the ADV locus, is associated with a wider set of AJ genes, located in the center of the AJ locus. Taken together, our results indicate that, in addition to the progressive opening of the ADV and AJ loci, the chromosomal location of ADV and AJ genes is a factor affecting AJ use in BALB/c mice.