The lymphocytes which infiltrate tumours and are grown in vitro to be used in adoptive immunotherapy are often characterized by dominant rearrangement of their T cell receptor (TCR) genes. To investigate the frequency and function of cells contributing to the 'dominant' rearrangement, we have cloned two bulk cell lines of TIL derived from melanoma patients (TIL-1 and TIL-5). These IL-2-propagated TIL cell lines had a CD8+ phenotype and exerted strong cytotoxic activity against autologous melanoma cells, but not against the natural killer (NK)-sensitive K-562 cell line or LAK targets such as Daudi cells. We derived 40 clones from TIL-1 and 23 from TIL-5. All tested clones were CD3+, CD4-, CD8+ and expressed the alpha/beta TCR. From TIL-1, 27 of 40 clones, and 13/19 of the TIL-5 clones lysed autologous tumour cells. In contrast to the NK-negative bulk cultures, K-562 killing was detected in 21 of the TIL-1 clones and 17 of the TIL-5 clones. TIL-1 contained eight clones and TIL-5 two clones with lytic capacity against neither autologous tumour cells nor the K562 cell line, although these clones possessed lytic potential as evidenced in a lectin-mediated lysis assay. LAK activity was not detected in most clones. Cytotoxic activity against autologous tumour could be inhibited by preincubation with anti-CD3 or anti-HLA class I MoAbs. Of the 34 TIL-1 clones analysed, 15 shared a rearranged TCR beta EcoRI restriction fragment of approximately 9.5 kb with the bulk culture. Clones sharing the EcoRI 10.5-kb dominant band present in TIL-5 bulk culture were also isolated. When the pattern of TCR beta rearrangement was compared with the cytotoxic functions, the following conclusions could be drawn: (i) clones contributing to the dominant band had heterogeneous functions. Most killed autologous tumour cells, but clones with no cytotoxic activity or even with no proliferative capacity in response to autologous tumour cells were also detected among those contributing rearrangement; (ii) some clones that share an apparently identical rearranged band different from the 'dominant' rearrangement, may demonstrate the same cytotoxic function. In addition, our data suggest that many of the clones that share the dominant rearrangement originated from diverse progenitors. The high frequency of clonally diverse anti-tumour reactive TIL is likely to be a reflection of the in vivo selection of the TCR repertoire at the site of tumour.(ABSTRACT TRUNCATED AT 400 WORDS)