Liver injury in murine chronic graft-vs-host disease (CGVHD) to minor histocompatibility Ag, B10.D2----BALB/c (600 rad), is characterized by mononuclear cell inflammation and necrosis of interlobular bile ducts. Bile duct destruction in this model is similar to that which occurs in human CGVHD, late liver transplant rejection, and primary biliary cirrhosis. This model provides a unique opportunity to isolate mononuclear inflammatory cells from the liver during CGVHD, study their functions, and investigate the immunologic mechanisms responsible for bile duct destruction. In the present study, we compared the in vivo organ homing of mononuclear inflammatory cells (MC) isolated from the liver and spleen during the course of CGVHD. MC isolated from the liver showed a progressive increase in homing to the livers of BALB/c mice from day 7 through 42. In contrast, the hepatic homing of MC isolated from the spleen peaked at day 21 and subsequently declined. CGVHD spleen MC showed a progressive increase in homing to the spleen of BALB/c mice whereas CGVHD liver MC showed no change over time. Homing to other organs was negligible. The hepatic and splenic homing of MC isolated during CGVHD was significantly greater in BALB/c (host) mice than in B10.D2 (donor) mice. Autoradiography was used to determine the intrahepatic sites at which CGVHD liver MC accumulate after i.v. injection into BALB/c mice. The results indicated that MC isolated from the liver when bile duct inflammation is most intense accumulate preferentially in hepatic portal spaces in close proximity to interlobular bile ducts. These results suggest that hepatic homing by CGVHD liver MC is specific for minor histocompatibility Ag expressed on host biliary epithelial cells. These data support the hypothesis that bile duct destruction in murine CGVHD is mediated by MC that are sensitized to minor histocompatibility Ag expressed by host biliary epithelial cells.