CTLs that recognize tumor Ags have been described in mice and humans, particularly for melanoma. These CTLs are CD8+, which is MHC-restricted. In contrast, in human carcinomas of the breast, pancreas, or ovary, and in multiple myeloma, CD8+ CTLs have been described that lyse targets expressing human MUC1 in a non-MHC-restricted manner. On the basis of these observations, we immunized mice with conjugates of mannan-human fusion protein, human mucin 1 (MUC1), which produced CD8+ CTLs. In contrast to the human anti-MUC1 CTLs found in cancer patients, the murine anti-MUC1 CTLs were clearly MHC-restricted, e.g., in inbred mice of the H-2-b, d, k, s, or z haplotypes; the H-2 restriction was also confirmed in H-2 congenic strains. Tests of H-2 recombinant strains demonstrated that MUC1 peptides were able to associate with D or K class I molecules of the b, d, or k haplotypes. Mice lacking MHC-class I molecules made weak CTL responses that were H-2Db-restricted, and in the class I H-2Kbm1 mutant strain, CTL restriction was also shown. Finally, cold target inhibition studies demonstrated that Kb and Db are recognized similarly, but Kk is less well recognized. Thus, anti-MUC1 CTLs induced by immunization of mice are different from those obtained from patients. The immunization of cancer patients with MUC1 peptides is now undergoing clinical trials and it will be of interest to observe whether the CTLs induced are HLA-restricted, not restricted, or whether both types of CTLs are produced.