Tumor specimens and ascites of patients with advanced ovarian cancer were utilized to obtain both primary ovarian carcinoma cell cultures and lymphocytes: tumor-infiltrating lymphocytes (TILs) from solid tumor tissue and tumor-associated lymphocytes (TALs) from peritoneal fluid. Tumor lymphocytes were grown in coculture with autologous tumor cells and recombinant human IL-2 (rhIL-2) for up to 4 weeks and at weekly intervals these were examined with respect to phenotype and cytotoxicity. The phenotype was studied using flow cytometry for a variety of human immunocompetent cell surface markers (CD3, CD4 CD8, CD16, CD56, TCR alphabeta, TCRgammadelta). Cytotoxicity was investigated using 4-hr 51Cr-release assays with the primary ovarian carcinoma cell cultures and the K562 cell line as target cells. The tumor lymphocytes did not demonstrate any obvious trend in phenotype changes during culture, although for different cultures a large range was noted for the various lymphocyte populations studied. Cytotoxicity against both autologous and allogeneic targets declined with culture length for the majority (6/7) of the lymphocyte cell lines tested (greatest at 1 week and least at 3 weeks). These initial results indicate that an in vitro non-MHC-restricted cytotoxic function of peritoneal lymphocytes can be effectively activated with IL-2 and autologous tumor cells. However, if activated lymphocytes are to be employed as a form of immunotherapy, they should be given within the first week of culture for maximum cytotoxic effect.