Chlamydia pneumoniae causes chronic infections, which have been associated with cardiovascular diseases. The antigenic structures of the organism have been detected in atherosclerotic lesions by immunohistochemistry. We wanted to further evaluate the presence and localization of C. pneumoniae in different tissues by in situ hybridization. We established a new colorimetric in situ hybridization method using a digoxigenin-labelled probe and studied the localization of C. pneumoniae in formalin-fixed, paraffin-embedded lungs of infected mice. We also used the method to study its presence in 12 abdominal aortic aneurysms. In C. pneumoniae-infected mice, the organism was first detected in bronchial epithelial cells, and later in pneumocytes and endothelial cells. C. pneumoniae was also present in five of eight abdominal aortic aneurysms previously shown to be positive by immunohistochemistry. The findings are in accordance with the invasive nature of C. pneumoniae, and confirm its presence in abdominal aortic aneurysms.