The T-cell receptor (TCR) alpha locus is thought to undergo multiple cycles of secondary rearrangements that maximize the generation of alphabeta T cells. Taking advantage of the nucleotide sequence of the human Valpha and Jalpha segments, we undertook a locus-wide analysis of TCRalpha gene rearrangements in human alphabeta T-cell clones. In most clones, ValphaJalpha rearrangements occurred on both homologous chromosomes and, remarkably, resulted in the use of two neighboring Jalpha segments. No such interallelic coincidence was found for the position of the two rearranged Valpha segments, and there was only a loose correlation between the 5' or 3' chromosomal position of the Valpha and Jalpha segments used in a given rearrangement. These observations question the occurrence of extensive rounds of secondary Valpha-->Jalpha rearrangements and of a coordinated and polarized usage of the Valpha and Jalpha libraries. Fluorescence in situ hybridization analysis of developing T cells in which TCRalpha rearrangements are taking place showed that the interallelic positional coincidence in Jalpha usage cannot be explained by the stable juxtaposition of homologous Jalpha clusters.