We previously introduced the concept that intrahepatic bile duct epithelial cells, or cholangiocytes, are functionally heterogeneous. This concept is based on the observation that secretin receptor (SR) gene expression and secretin-induced cAMP synthesis are present in cholangiocytes derived from large (>15 μm in diameter) but not small (<15 μm in diameter) bile ducts. In work reported here, we tested the hypothesis that cholangiocytes are heterogeneous with regard to proliferative capacity. We assessed cholangiocyte proliferation in vivo by measurement of [³H]thymidine incorporation and in vitro by both [³H]thymidine incorporation and H₃ histone gene expression in small ( fraction 1) and large ( fraction 2) cholangiocytes isolated from rats after bile duct ligation (BDL). In the two cholangiocyte subpopulations, we also studied basal somatostatin receptor (SSTR₂) gene expression as well as the effects of somatostatin on 1) SR gene expression and secretin-induced cAMP synthesis and2) [³H]thymidine incorporation and H₃ histone gene expression. In normal rat liver, cholangiocytes, unlike hepatocytes, were mitotically dormant; after BDL, incorporation of [³H]thymidine markedly increased in cholangiocytes but not hepatocytes. When subpopulations of cholangiocytes were isolated after BDL, DNA synthesis assessed by both techniques was limited to large cholangiocytes, as was SSTR₂ steady-state gene expression. In vitro, somatostatin inhibited SR gene expression and secretin-induced cAMP synthesis only in large cholangiocytes. Moreover, compared with no hormone, somatostatin inhibited DNA synthesis solely in large cholangiocytes. These results support the concept of the heterogeneity of cholangiocytes along the biliary tree, extend this concept to cholangiocyte proliferative activity, and imply that the proliferative compartment of cholangiocytes after BDL is located principally in the cholangiocytes lining large (>15 μm) bile ducts.