To assess the possible involvement of canine adenovirus type 1 (CAV-1) in naturally occurring cases of canine chronic liver disease, a polymerase chain reaction (PCR)-based assay was developed to detect a conserved region of the major core protein gene (pVII) of CAV-1 in formalin-fixed, paraffin-embedded liver sections. Results were compared with a standard avidin-biotin immunoperoxidase complex technique that detected CAV-1 antigens using a commercial monoclonal anti-adenovirus antibody. Seventeen cases of cirrhosis and 28 cases of chronic hepatitis with piecemeal necrosis and progressive fibrosis were selected for the study. Formalin-fixed, paraffin-embedded liver sections of 2 cases of infectious canine hepatitis (ICH) and crude DNA extract from CAV-1 (ATCC VR 293 Utrecht strain) served as positive controls. A 411-base-pair viral region was amplified and sequenced as CAV-1 pVII in both cases of infectious canine hepatitis and in the CAV-1 crude DNA extract. The 2 ICH cases were positive for CAV-1 antigens by the immunoperoxidase method. CAV-1 DNA or antigens were not detected by either technique in any of the 45 cases of chronic liver disease selected for the study. These results indicate that both PCR and immunohistochemistry are reliable and rapid techniques for detecting CAV-1 in formalin-fixed, paraffin-embedded liver sections of dogs with ICH. Several possibilities may explain the negative results obtained with both techniques in this study, including the noninvolvement of CAV-1 in canine chronic hepatitis and cirrhosis and the possibility that the virus causes initial damage, provokes a self-perpetuating chronic liver disease, and disappears.