Sixteen metastatic breast cancer patients were immunized with a low dose (5 micrograms) of a 16 amino acid MUC1 peptide (GVTSAPDTRPAPGSTA) conjugated to KLH (BP16-KLH) plus DETOX adjuvant and evaluated for antibody titers against MUC1 peptide and KLH and for cytotoxic lymphocyte (CTL) activity using class 1 HLA-matched MUC1-positive tumor targets. All patients generated strong anti-KLH IgG responses. Only 3 patients developed an anti-MUC1 IgG response, which was weak in magnitude. As it is controversial whether human cancer patients generate class-1-restricted CTL against MUC1, we examined anti-MUC1 CTL activity of PBLs following 4 immunizations with BP16-KLH. The generation of MUC1-specific CTLs required only a 6-day in vitro stimulation of patients' T-cells with synthetic MUC1-peptide-pulsed autologous APCs. The assay for CTL activity was a 4 hour 51Cr release from labeled adenocarcinoma target cells. Eleven of the 16 immunized patients were tested for CTL activity using class-1-matched adenocarcinoma target cell lines. Evidence for class-1-restricted killing of MUC1-expressing tumor cell lines was obtained in 7 of these 11 patients.