In adenocarcinomas of the breast and pancreas, underglycosylation of the glycoprotein MUC1, also expressed by normal breast and pancreatic ductal epithelial cells, results in new protein epitopes to which the immune system mounts a cytotoxic T cell response. This cytotoxic immune response is directed primarily against epitopes on the tandem repeat domain of MUC1, and is unconventional in that it is major histocompatibility complex (MHC)-unrestricted. It is therefore necessary to investigate the molecular basis of this immune response in order to enhance and optimize it for immune therapy purposes. In the present study, we characterize new MUC1 transfected human lymphoblastoid cell lines C1R and T2, and a pig kidney epithelial line LLC-PKI, that express MUC1 with either two repeats (MUC1-2R) or 22 repeats (MUC1-22R), and use them as stimulators and targets for cytotoxic T cells (CTL) in vitro. We show that MUC1-2R is processed and glycosylated similarly to MUC1-22R. In contrast to MUC1-22R, MUC1-2R is not recognized by CTL on T2 and C1R cells known for no or low MHC class I expression. It is however recognized when expressed at high density on xenogeneic LLC-PKI cells. We propose that in MHC-unrestricted recognition, a large number of MUC1 epitopes is necessary to effectively engage the T cell receptor, and that in the presence of a low number of epitopes, engagement of the CD8 co-receptor by MHC class I molecules may be required for completing the signal through the T cell receptor.