Tumor-infiltrating lymphocytes (TIL) from 16 squamous cell carcinomas of head and neck (SCCH&N) and four nonsquamous cell carcinomas were studied. By immunoperoxidase staining in situ, the tumors studied were found to be infiltrated mainly by CD2+CD3+ cells, and 30-50% of the T-lymphocytes were HLA-DR positive and transferrin-receptor positive. They also contained scarce NKH1+ cells. When TIL as well as autologous peripheral blood lymphocytes (A-PBL) were cultured in 1,000 U/ml of recombinant interleukin 2 (rIL2), TIL proliferated in all but three cases, and A-PBL proliferated in all but two cases. Frequently, but not always, TIL expanded better than A-PBL. The median expansion for TIL was 100-fold and that for A-PBL was 31-fold in long-term cultures maintained for up to 88 days. TIL obtained from untreated primary SCCH&N were initially delayed for up to 20 days in their proliferative response to rIL2, but then grew well. In contrast, TIL and A-PBL from metastatic SCCH&N either did not proliferate or were delayed in their proliferative response for up to 40 or 50 days. A-PBL, when tested early (days 10-20 in culture), showed the highest cytotoxic activity against cultured and fresh tumor-cell targets, whereas TIL were most active later in culture (days 20-30). On a per culture basis, TIL achieved higher antitumor cytotoxicity than A-PBL. By day 80, lytic activities of most TIL cultures declined to undetectable levels. CD3+Leu19- T-lymphocytes were the major expanding cell population in most TIL cultures. However, these cells were poor mediators of antitumor cytotoxicity in TIL or A-PBL cultures as shown in cell sorting experiments. The antitumor effector cells expressed CD3-Leu19+ and/or CD3+Leu19+ phenotypes. On Giemsa-stained smears, these two types of IL2-expanded effector cells had the morphology of large granular lymphocytes. Our results indicate that TIL from human SCCH&N could be expanded and reach high levels of antitumor effector function in long-term cultures with rIL2.