Most T-lymphocytes express a highly specific antigen receptor (TCR) on their cell surface, consisting of a clonotypic alphabeta-heterodimer. Both alpha- and beta chains are products of somatic rearrangements of V, (D) and J gene segments encoded on the respective loci. The qualitative, quantitative and dynamic aspects of the TCRalpha chain repertoire of humans and mice have been difficult to estimate, mainly due to locus complexity. Analyses of the T-cell repertoire were first performed at the transcriptional level using classical cloning and sequencing strategies and then later at the genomic level using sensitive multiplex PCR assays that allow surveying the global rearrangement of the TCRAD locus. These all converge and support the conclusion that the V-J recombination pattern in both human and mouse thymus is not random but depends on the reciprocal V and J positions within the locus, thereby limiting the combinatorial diversity of the TCRalpha chain repertoire. The recombination profile is compatible with a sequential opening of the V region with progressive tracking along the two regions in opposite directions starting from the nearest and then moving towards the most distant V and J gene segments. In this chapter, we report new insights into the degree of human and mouse TCRalpha chain diversity in thymic and peripheral T-lymphocytes. Since the comparison of human and mouse V-J recombination shows a similar pattern of rearrangement, we suggest that spatial and temporal synchronization on the accessibility of V and J gene segments are general features of V-J rearrangements that are conserved throughout evolution.