The influence of the epithelial mucin MUC1 on T cell-mediated lysis was analysed using lymph node lymphocytes (LNL) from patients with colorectal carcinoma. LNL were stimulated with allogeneic, MUC1-transfected B cells and the bulk cultures were cloned. Alloreactive cytotoxic T cell clones were obtained which preferentially lysed MUC1-expressing targets. The majority was CD4+ and MHC-class II-restricted, and a minor group was CD8+ and MHC-class I-restricted. All the clones expressed CD3 and TCR alpha beta, and were CD56-. The capacity to preferentially kill MUC1-expressing targets was stable in several clones for up to 6 months in culture. The enhancing effect of MUC1 on the lysis was investigated in more detail. It was only seen after inhibition of O-linked glycosylation in the targets. Furthermore, this effect was completely abrogated by the monoclonal antibody 3C9, directed against the Thomsen-Friedenreich antigen (T-antigen, Gal beta 1-3GalNAc bound alpha 1-3 to Ser/Thr) as well as by the soluble disaccharide Gal beta 1-3GalNAc, but not by other similar disaccharides. The authors conclude that in their system the preferential killing of MUC1-expressing targets is due to the recognition of an internal carbohydrate epitope accessible on under-glycosylated MUC1, possibly T-antigen, by an auxiliary receptor molecule on T cells.