MUC1 was first defined as a tumor antigen in the late 1980s, yet little is known about the types of immune responses that mediate rejection of MUC1(+) tumors in vivo. MUC1-specific antibodies, T(h) cells and cytotoxic T cells can be detected in patients with different adenocarcinomas, yet these tumors usually progress. Thus, there is a need to better understand the in vivo mechanisms of antigen-specific tumor rejection. To characterize the nature of MUC1-specific immune responses in vivo, rejection of a MUC1-expressing melanoma tumor line (B16.MUC1) was evaluated in mice lacking specific T cell subsets, cytokines, co-stimulatory molecules or molecular effectors of cytolytic pathways. Results demonstrated that rejection of the B16.MUC1 tumor cell line was primarily mediated by CD4(+) T cells, and required Fas ligand, lymphotoxin-alpha, CD40, CD40 ligand and CD28, but not perforin, gammadelta T cells, IL-4, IL-10, IL-12 or tumor necrosis factor receptor-1. Depletion of NK cells demonstrated that NK cells might also contribute to MUC1 immunity in the B16.MUC1 tumor model. These results demonstrated that the immune response generated against MUC1 does not fit the type 1 or 2 model described for many immune responses. Additionally, multiple cytolytic mechanisms are required for B16.MUC1 rejection.