We have previously reported that tumor-specific cytotoxic T lymphocytes (CTLs) derived from pancreatic and breast cancer patients recognize specific epitopes on the mucin polypeptide core. These CTLs recognize breast and pancreatic tumor cells in a major histocompatibility complex (MHC)-unrestricted fashion, and the lytic activity of these T cells is mediated through the T cell receptor (TCR). To characterize the TCR-mediated MHC-unrestricted CTL function, we used semiquantitative polymerase chain reaction (PCR) and cytofluorometry to analyze the TCR repertoire in CTL lines established from cancer patients and specific for mucin-expressing tumors. We found three TCR Vbeta genes, Vbeta9, Vbeta13.1. and Vbeta17, predominantly expressed in these functional cell lines, established either from one patient by stimulation with various mucin-expressing targets or from different patients. Sequencing of these preferentially used TCR genes unveiled usage of distinct Jbeta and Cbeta but a potentially interesting conservation of certain amino acids in the CDR3 region.