Tumor infiltrating lymphocytes (TIL) isolated from 12 patients with metastatic malignant melanoma, renal cell carcinoma, or breast adenocarcinoma were expanded in rIL-2 for 22 to 45 days (median 33 days) and analyzed for lymphokine mRNA expression and patterns of TCR gene rearrangement. All TIL cultures were significantly enriched for T cells, with CD3+ CD8+ cells predominant in 8 of 10 cases tested, and demonstrated an oligoclonal (rather than polyclonal) pattern of TCR gene rearrangement. Nine of 12 cultures could effectively lyse the autologous targets in short term chromium release assays. IL-2 expanded-TIL expressed mRNA for TNF-alpha and TNF-beta (lymphotoxin) and, in 5 of 9 (41%) cases, granulocyte/macrophage-colony stimulating factor mRNA but not IL-1 beta or IL-2 transcripts. Cultured TIL deprived of rIL-2 for 4 days did not constitutively express mRNA for any of the lymphokines tested. One long term TIL line in culture was followed and periodically tested for lytic activity and TNF-mRNA expression. Loss of the specific cytolytic but not proliferative activity at day 85 was associated with disappearance of TNF mRNA. Profiles of lymphokine secretion may provide a useful marker for functionally characterizing different T cell subsets and may provide correlates of the in vivo anti-tumor effects of these cells when TIL are adoptively transferred into cancer-bearing patients.