Mice transgenic for the human MUC1 carcinoma‐associated antigen (MUC1.Tg) are tolerant to immunization with MUC1 antigen. Recent studies, however, have demonstrated that immunization of MUC1.Tg mice with fusions of MUC1‐positive tumour and dendritic cells (FC/MUC1) reverses MUC1 unresponsiveness and results in rejection of established MUC1‐positive pulmonary metastases. Here we demonstrate that lymph node cells from MUC1.Tg mice immunized with the FC/MUC1 fusion cells proliferate in response to MUC1 antigen by a mechanism dependent on the function of CD4, major histocompatibility complex (MHC) class II, B7‐1, B7‐2, CD28, CD40 and CD40 ligand. The findings demonstrate that stimulation of lymph node cells with MUC1 results in selection of MUC1‐specific CD8⁺ T cells. We show that the CD8⁺ T cells exhibit MUC1‐specific cytotoxic T lymphocyte (CTL) activity by recognition of MUC1 peptides presented in the context of MHC class I molecules K^b and D^b. The MUC1‐specific CD8⁺ T cells also exhibit antitumour activity against MUC1‐positive metastases, but with no apparent reactivity against normal tissues. These results indicate that immunization of MUC1.Tg mice with FC/MUC1 reverses immunological unresponsiveness to MUC1 by presentation of MUC1 peptides in the presence of costimulatory signals and generates MHC‐restricted MUC1‐specific CD8⁺ T cells.