From the primary site of a pancreatic adenocarcinoma (patient BE) a permanent cell line (MZ-PC-2) was established in tissue culture. In the course of mixed lymphocyte-tumor-cell cultures (MLTC) with autologous blood-derived lymphocytes, we isolated CTL clones that lysed autologous tumor cells but not autologous EBV-transformed B cells (EBV-B) and not K562. Pre-treatment of MZ-PC-2 cells with IFN-gamma was required to obtain significant lysis in 4-hr cytotoxicity assays. IFN-gamma was superior to IFN-alpha in that respect. Among MLTC responder lymphocytes, tumor-reactive CTL proliferated more strongly in response to MZ-PC-2 cells treated with IFN-gamma than to untreated tumor cells. Three CTL clones derived from MLTC were chosen for further analysis. They were CD3+, CD8+, TCR-alpha/beta+ and behaved identically in all functional aspects tested. They all expressed the same TCR-beta chain, indicating that they descended from a common precursor lymphocyte and were directed against the same antigen. According to antibody-inhibition experiments, BE-CTL recognized their targets via an HLA-B molecule carrying the Bw6 supertypic determinant. Irrespective of pre-incubation with IFN-gamma, low levels of tumor-cell lysis, or none, were seen when MZ-PC-2 cells were kept in medium supplemented with autologous serum or serum pooled from healthy volunteers instead of FCS. Lysability was restored when TNF-alpha was added to human serum. Serum-free medium was found to enhance the susceptibility of MZ-PC-2 cells to lysis by autologous CTL.