MUC1 is a tumor-associated antigen that is overexpressed in invasive ductal carcinomas of the pancreas (PC). MUC1-specific cytotoxic T lymphocytes (CTLs) recognize MUC1 molecules in a HLA-unrestricted manner. In this study, we performed adoptive immunotherapy (AIT) in patients with PC with CTLs stimulated by the MUC1-expressing human PC cell line YPK-1. To induce CTLs, peripheral blood mononuclear cells (PBMCs) were cultured for 3 days with inactivated YPK-1 cells and then stimulated with interleukin (IL)-2 for 7 days. The cytotoxicity of these cells against human cancer cell lines was analyzed, and a variety of antibodies were evaluated for their ability to inhibit cytotoxicity. We treated 8 patients with unresectable PC and 20 patients with resectable PC postsurgically. CTLs were induced as described above, suspended in 100 ml saline and injected intravenously. Induced CTLs were cytotoxic against 5 MUC1-expressing PC cell lines and a breast cancer cell line, regardless of the HLA phenotype. Low cytotoxicity was observed in 7 MUC1-negative cancer cell lines. Anti-CD3 monoclonal antibody (mAb) or anti-CD8 mAb strongly inhibited cytotoxicity against YPK-1, whereas anti-class I mAb showed no inhibition. YPK-1 cells incubated with anti-MUC1 mAb also showed low cytotoxicity. Clinically, the median survival time was 5.0 months for patients with unresectable PC treated with AIT. None of the 5 patients without liver metastasis showed hepatic recurrence. The median survival time was 17.8 months for 18 out of 20 patients with resectable PC who underwent curative surgery, and the 1-, 2- and 3-year survival rates after surgery were 83.3, 32.4, and 19.4%, respectively. Liver metastasis was found in only one patient and no side effects of AIT were observed. CTLs stimulated by a MUC1-expressing human pancreatic cancer cell line showed a strong tumor cytotoxic activity in a MUC1-specific and MHC-unrestricted manner. AIT with stimulated CTLs significantly suppressed the postsurgical hepatic recurrence of PC. Adjuvant immunotherapy with CTLs may be useful in the postsurgical treatment of PC.