We characterized tumor-infiltrating lymphocytes (TIL) from ascites of patients with ovarian or pancreatic cancer in which the human tumor necrosis factor (TNF) gene was successfully transduced with retrovirus vector. The TNF-gene-transduced TIL (TNF-TIL) from these patients showed a higher level of TNF production and higher cytotoxic activity against K562 and Daudi cells than did neomycin-phosphotransferase-gene-transduced TIL (neo-TIL). Of these TIL preparations, only that from pancreatic cancer was further characterized since it was collected in a relatively large amount. In spite of the fact that the autologous tumor cells showed resistance to soluble TNF, the TNF-TIL clearly demonstrated enhanced cytotoxicity against them as compared with neo-TIL. The enhanced cytotoxicity was ascribed to autocrine effects of secreted TNF on TIL, which included augmentation of adhesion molecule (CD2 and CD11a) and interleukin-2 receptor expression, and elevation of production of interferon gamma, lymphotoxin and granulocyte/macrophage-colony-stimulating factor and its paracrine effect on target cells to facilitate them to be more susceptible to TIL.