Certain T cell antigen receptor V gene products in man have been shown by us and others to display a reproducible bias for preferential expression in CD4+ or CD8+ T cell subsets. In order to investigate whether such a skewed representation of V gene segments is also present at the J gene segment level, we tested the relative Jβ gene usage by Vβ 5.1+ T cells, as this Vβ gene is biased towards CD4+ T cell expression in virtually all individuals. To analyze the usage of the 13 Jβ gene segments, we developed a new approach using Vβ 5.1 and Cβ specific oligonucleotides as 5‘ and 3’ primers respectively for polymerase chain reaction (PCR) amplification of cDNA derived from CD4+ or CD8+ peripheral blood lymphocyte (PBL) T cells. The PCR products were visualized for reactivity with individual Jβ 1.1 –1.6 and Jβ 2.1–2.7 32P-labelled oligonucleotide probes using autoradiography and quantitative gel-scanning. Eleven normal blood donors provided the PBL T cells. The results showed that in every individual‘s Vβ 5.1+ T cell populations (CD4 and CD8), all Vβ /Jβ combinations were used although at varying but reproducible levels for each J0 gene. Thus, no discernible disallowance of combinations existed. Moreover, we could show that six of 13 Jβ genes were unequally expressed when compared in pairs with regard to expression in CD4+ and CD8+ T cell subsets. The Jβ 1.3, 1.4, 1.6, 2.5 and 2.6 gene segments showed a biased representation towards CD4+ T cells, while the Jβ 2.7 gene segment was expressed more frequently by CD8+ T cells. We conclude that we have developed a simple and reproducible assay allowing the quantitative determination of individual Jβ gene usage in relation to individual V genes in CD4/CD8 T cell subsets. In addition, one individual with an unusually high expression of peripheral Vβ 5.1+ CD4+ T cells (9.9%) showed an exceptionally high Jβ 2.1 gene segment usage. This finding indicates the sensitivity and usefulness of the experimental approach to detect even minor clonal expansions in complex cell populations.