We attempted to detect chlamydial antigens in canine atherosclerotic lesions from seven dogs by immunohistochemical technique using anti-Chlamydia psittaci (C. psittaci) polyclonal and anti-C. pneumoniae monoclonal antibodies. Immunopositive signals to both antibodies were recognized in the atherosclerotic lesions of the aortas, coronary and splenic arteries of all dogs. Positive signals were found in the foamy cytoplasm of infiltrated macrophages and extracellular matrices in the lesions. In some lesions, cytoplasm of the endothelial cells and smooth muscle cells was also immunopositive against both antibodies. By electron microscopy, chlamydial microorganisms were found in the cytoplasm of endothelial cells and smooth muscle cells. Using polymerase chain reaction (PCR), detection of C. pneumoniae DNAs were performed in the spleen, heart (coronary arteries) and kidney in one of the seven dogs. Positive 314 bp PCR products were obtained in all samples of the dog. These results confirmed the presence of viable Chlamydiae in atheromas and supported the conclusion that the organism may be an active factor in the pathogenesis of canine, as well as human atherosclerosis.