Neuroblastoma (NB) is a major-histocompatibility-complex(MHC)-negative neuroectodermal tumour that is often infiltrated with lymphocytes. A detailed characterization of NB-associated tumour-infiltrating lymphocytes (TIL) has never been carried out. Here we have investigated the immunophenotype and the cytotoxic activities of TIL from nine and seven NB patients respectively. Furthermore, the T cell receptor (TcR) variability and the patterns of cytokine gene expression of fresh versus recombinant (r) interleukin(IL)-2-cultured TIL were studied in four NB cases. The results obtained showed the following: (1) freshly isolated TIL were comprised of a mixture of CD4⁺ and CD8⁺ T cells partially expressing HLA-DR and/or CD25. The CD4/CD8 ratio ranged from 0.5 to 5 in the different cases. Upon culture of TIL with rIL-2, an increased proportion of CD56⁺ and CD8⁺ lymphocytes was consistently observed; (2) IL-2-expanded TIL lysed natural-killer(NK)-sensitive and lymphokine-activated-killer (LAK)-sensitive target cell lines; (3) reverse-transcriptase/polymerase-chain-reaction (RT-PCR) experiments showed that most TcR Vβ genes were expressed both in fresh and in cultured TIL, suggesting that such cell populations were polyclonal; (4) interferon γ, IL-4, IL-5, tumour necrosis factor (TNF) α, IL-8, IL-10 mRNA and, to a lesser extent, IL-2 mRNA were expressed by cultured TIL, as assessed by RT-PCR; the corresponding tumour samples consistently contained TNFα, IL-8 and IL-10 mRNA, whereas IL-2 and IFNγ mRNA were faintly expressed in some NB tumors and IL-4 and IL-5 mRNA were never detected. A total of 90 clones were subsequently raised from IL-2-expanded TIL from six NB patients; 87/90 clones were of T cell lineage with a CD4⁺ or CD8⁺ immunophenotype, whereas the 3 remaining clones were of NK cell origin. Upon triggering of the CD3-TcR complex, 64% CD4⁺ and 77% CD8⁺ T cell clones killed the murine P815 mastocytoma cell line. Virtually no T cell clone lysed a LAK-sensitive NB cell line, whereas 15% CD4⁺ and 17% CD8⁺ clones mediated NK-like activity against the K562 cell line. Finally, the patterns of cytokine production by CD4⁺ clones were roughly consistent with those of a T helper (Th)1 profile and similar to those observed in CD8⁺ clones.