We have investigated whether or not polysaccharide preparation PSK directly augments the proliferation and cytotoxicity of tumor-infiltrating lymphocytes (TILs). TILs were separated from 10 patients with gastrointestinal cancer (5 gastric cancers, 3 colon cancers and 2 pancreatic cancers). TILs were cultured with IL-2 and PSK for 7 days. The DNA synthesis of TILs was augmented by incubation with 100 micrograms/ml of PSK, which was similar to serum level with oral administration of PSK in cancer patients. The effect of PSK in DNA synthesis was also found by elimination of non-T cells. Furthermore, we established TIL clones and examined the effect of PSK on TILs clones. The DNA synthesis was augmented by PSK in CD4 positive and CD8 positive TIL clones without non-T cells, suggesting that PSK acts directly on TILs. We examined the cytotoxic activities of TILs by the 4-h and 16-h 51Cr release assay. PSK did not affect the cytotoxic activity of TILs against autologous tumor cells and KATO-III cells in the 4h 51Cr release assay, whereas PSK induced high lysability of TILs against autologous tumor cells in the 16-h 51Cr release assay. We studied the ability of PSK to induce cytokines from TILs using a double chamber plate. The DNA synthesis of tumor cells was more suppressed by the mixed-tumor cell culture supernatants of TILs cultured with PSK, compared to that of TILs cultured without PSK. It is suggesting that PSK induced long term killing activity of TILs by induction of cytotoxic cytokines. Thus, PSK augmented the proliferative response of TILs without interaction of T cells and non-T cells and induced cytotoxic cytokines of TILs.