A strategy, based on using V beta family-specific oligonucleotides, was developed for specific amplification and direct sequencing of human TCR V beta genes. With this strategy, it was possible to undertake a structural analysis of TCRs from human T cell clones in specific responses. 12 HLA-B27-specific cytotoxic clones were examined. The results reveal a nonrandom use of V beta gene diversity in this alloreactive response in that: (a) the clones express a restricted number of V beta segments, including a subset of V beta families that are significantly more related to one another than to most other V beta families; (b) five of seven clones having a particular reaction pattern with HLA-B27 subtypes possess Alanine at the D-J junction; and (c) identical J beta segments are found associated in several instances with identical or highly homologous V beta gene segments. In addition, two new V beta 13 members are reported.