As part of a phase-II clinical trial of post-operative active specific immunization (ASI) with virus-modified autologous tumour cells (AuTu) in colorectal carcinoma patients, we have analyzed in vitro anti-AuTu immune responses with lymphocytes isolated from the peripheral blood (PBL) of 5 treated patients. The PBL of 3 "responder patients", those who developed a positive DTH reaction to AuTu, when stimulated in standard in vitro autologous lymphocyte tumour-cell cultures (ALTC), showed cytotoxic anti-AuTu reactivity only in association with natural-killer-cell(NK)-like activity. We removed nonspecific cytotoxic cells (CD56-positive) from PBL of colon carcinoma or melanoma patients and positively selected T cells with strong CD8 staining (CD8hi) using FACS. Following in vitro stimulation, specific cytotoxic T cells (CTL) directed against either autologous EBV-transformed B cells (AuEBV-B) or autologous melanoma cells were identified in the CD8hi T-cell population. However, even using this novel technique, no specific CTL against autologous colon carcinoma cell lines were detected in PBL from ASI-treated patients (2 DTH responders and 2 DTH non-responders). If AuTu-specific CTL precursors existed in these blood samples, their frequency must have been very low (less than 1 in 8 x 10(4) CD8 positive T cells). Sorted CD4 T cells from these patients, in the presence of autologous antigen-presenting cells, showed no specific anti-tumour proliferative response, and in one instance we observed inhibition of proliferation in the presence of tumour cells.