A method was described for the generation of cells from tumor-bearing mice; these cells were capable of exhibiting significant antitumor reactivity when adoptively transferred into tumor-bearing hosts. Tumor cell suspensions from a variety of tumors were able to be separated using enzymatic techniques and they were cultured in medium containing recombinant interleukin-2. Activated infiltrating lymphocytes within these tumors expanded; and, by 6-8 days after initiation of culture, lymphocytes predominated and were able to grow to large numbers. The adoptive transfer of these tumor-infiltrating lymphocytes (TILs) made possible mediation of the reduction of established 3-day pulmonary micrometastases from 5 of 7 tumors tested, including two 3-methylcholanthrene (CAS: 56-49-5)-induced sarcomas, one 1,2-dimethylhydrazine (CAS: 540-73-8)-induced colon carcinoma, and the B16 melanoma, all in C57BL/6 mice, as well as the 1660 bladder carcinoma in BALB/c mice. Approximately 2-4 X 10(6) transferred cells were capable of totally eliminating 3-day established metastases. These cells were thus 50 to 100 times more effective than lymphokine-activated killer cells in reducing established metastases; however, they could not be generated from all tumors. The concomitant administration of recombinant interleukin-2 enhanced, by approximately fivefold, the in vivo activity of these cells. The specificity of action of TILs in vivo was different from that determined by classic amputation rechallenge experiments. The tumor-infiltrating lymphocytes that developed this antitumor reactivity appeared to be Thy-1+ and did not bear the asialo GM1 antigen. The potent antitumor effect of these TILs, when transferred in vivo to tumor-bearing hosts, raises the possibility of utilizing similar approaches for the isolation and therapeutic use of lymphocytes with antitumor reactivity from human tumors.