The phenotype of T cells associated with the common bile duct (CBD) is unknown. We investigated the hypothesis that they behave like other intraepithelial lymphocytes (IEL). Thus, we sought to determine the phenotype, TCR repertoire, and epithelial recognition of T cells obtained during endoscopic retrograde cholangiopancreatography. Three subjects were studied: two with primary sclerosing cholangitis and one normal control. After establishing a short-term T cell line, cells were 1) stained with mAbs for flow cytometric analysis, 2) analyzed for TCRB chain transcript expression, and 3) used as effector cells for cytotoxicity and proliferation. Flow cytometry revealed that for all the subjects 98% of the T cells were TCR-alpha beta-positive. Immunohistology of the CBD showed that the epithelium and lamina propria contained significant numbers of CD3+ CD43+ CD45RO+ lymphocytes. Complementarity-determining region 3 length displays suggested that the CBD-derived lines were oligoclonal. This was confirmed by cloning and random sequencing of PCR amplification products using TCRBV region family-specific primers; TCRB chain sequences were reiterated in all transcripts analyzed. In one case, two expanded TCRB clones could be identified that were persistent in the bile duct over a 1-yr period. The CBD-derived lines were cytolytic in a redirected lysis assay and caused cytolysis of an intestinal epithelial cell line (Caco-2). This recognition was likely preferential for intestinal epithelial cells, since a CBD-derived line exhibited proliferation to two intestinal epithelial cell lines (HT-29 and Caco-2) but not three other lines (HepG2, human foreskin fibroblast, and KD). We conclude that the CBD contains IELs that share several characteristics with intestinal IELs.