Optimal conditions for expanding tumor-infiltrating lymphocytes (TILs) specifically cytotoxic for autologous melanoma for clinical use have not yet been identified. In several small studies, interleukin (IL)-4 was reported to promote the growth of such TILs in IL-2. Given the potential implications for TIL therapy, we attempted to confirm these findings in a larger study. Baseline data were first obtained on the proliferation, immunophenotype, and cytotoxic reactivity to autologous melanoma of TILs cultured in IL-2 alone. Similar studies were performed with TIL cultured concurrently in either IL-2 alone or in a combination of IL-2 and IL-4. TILs were obtained by excisional biopsy of tumors from 52 patients with metastatic malignant melanoma; TILs from 38 patients were expanded in IL-2 (1,000 U/ml). TILs from 19 biopsies were maximally expanded 6- to 24,000-fold (median, 300-fold) over 4-10 weeks. Expansion did not correlate with the weight of, or number of lymphocytes in, the biopsy specimen, or the site of the biopsy (lymph node vs. subcutaneous metastases). During weeks 5-8, TILs from 19 of 25 biopsy specimens lysed autologous melanoma with little or no lysis of allogeneic melanoma. Lysis of autologous tumor was blocked by antibody to class I antigens. Twenty-four TIL specimens were cultured concurrently in IL-2 alone and in IL-2 plus IL-4 and tested for growth and for lysis of autologous and allogeneic melanomas. Although in five of 24 cultures TIL expansion was greater in the combination of IL-2 plus IL-4 than in IL-2 alone, in 17 of 24 cultures significantly less expansion was observed in the combination than in IL-2 alone (p - 0.037). TILs cultured in IL-2 plus IL-4 displayed enhanced lysis for autologous melanoma in only three of 19 TIL specimens tested, but were less lytic for autologous melanoma in 13 of 19 TIL specimens when compared to TILs grown in IL-2 alone (p=0.01). The results show that TILs with preferential cytotoxic reactivity against autologous melanoma are likely to grow better in IL-2 alone than in IL-2 plus IL-4. Therefore, IL-4 should not be used for generating TILs for clinical trials of adoptive immunotherapy of metastatic melanoma.