Tumor-infiltrating lymphocytes (TILs) from colorectal cancers were separated from tumor cells by enzymatic and mechanical tissue disaggregation and discontinuous density gradients. Peripheral blood lymphocytes (PBLs) were isolated using the same procedure. The freshly separated TILs and PBLs were analyzed phenotypically by flow cytometry. The CD4+/CD8+ ratios of the freshly isolated TILs and PBLs were comparable (> 1 in both lymphocyte populations). CD25+, HLA-DR+ and CD56+ cells were significantly more frequent in the TIL than in the PBL population. However, the number of CD45RA+ cells was lower in the TILs as compared to PBLs, while CD29+ accumulated by about 90% in TILs. TILs and autologous PBLs were expanded in vitro with rIL-2. The mean rate of proliferation after 4 weeks was 642-fold in TIL cultures and 335-fold in PBLs. More than 90% of the rIL-2-expanded lymphocytes expressed CD2 and the great majority was CD29+. Stimulation with rIL-2 in vitro induced an outgrowth of CD56+ cells mainly in the TILs. Accordingly the expression of CD3+ and alpha/beta receptor in the TILs was low. Those cells which phenotypically represented lymphokine-activated killer cells displayed a lytic activity against the autologous tumor as well as against allogeneic K562 and Daudi targets. In accordance with the better proliferative response of TILs in long-term cultures with rIL-2, the lytic activity of TILs against autologous and allogeneic tumor targets was significantly higher as compared to PBLs.