Tumor infiltrating lymohocytes (TIL) were isolated from various human cancers and cultured. The expansion, cytotoxicity, phenotype, and perforin, which is considered the cytotoxic factor of killer cells, of TIL were examinel. TIL cultured in 1000u/ml recombinant interleukin-2 (rIL-2) with 20% conditioned medium (CoM) exhibited far better expansion capability than that of TIL cultured in rIL-2 1000u/ml alone, and continued to expand up to 14 weeks. The maximal expansion index (EI) was 23912. When TIL arrested expansion during the culture, the addition of PHA restored the ability to expand. The CoM was composed of spent medium from a 3-day culture of peripheral blood mononuclear cells from cancer patients. The lymphokine-activated killer (LAK) cells induced by the culture was utilized for clinical adoptive immunotherapy in our department. Cytotoxicity against K562 and Daudi target cells was highest at 2 to 3 weeks, and disappeared between 4 and 7 weeks of culture. The phenotype of fresh TIL was mainly CD3(+) T cells, predominantly CD4(+) cells, but CD8(+) cells became predominant in long term culture. The frequency of IL-2R(+) and HLA-DR+ cells was correlated with the expansiveness of TIL. The appearance and intensity of perforin positive cells were associated with the level of cytotoxicity of cultured TIL.