Objectives Chlamydia pneumoniae has been related to atherosclerotic disease in both seroepidemiologic and genomic studies. We performed a case-control study to determine seropositivity and DNA detection in arteries of patients with peripheral artery occlusive disease and of healthy subjects. Methods The study included 64 patients with peripheral artery occlusive disease, and 50 control subjects who underwent varicose vein surgery, matched to the patient group for age, sex, and tobacco use. The fibrinogen level in all study subjects was measured as a marker of inflammation. Blood samples were taken from all subjects for determination of immunoglobulin (Ig) G elementary bodies (EB) against C pneumoniae with microimmunofluorescence (MIF) and enzyme-linked immunosorbent assay (ELISA), and of IgA EB with ELISA. The cutoff titers were 1:32 for MIF and 1.1 for ELISA. Biopsy specimens of arterial atheromatous plaque were obtained from patients, and of pudendal artery and saphenous vein from control subjects, and were studied with hemi-nested polymerase chain reaction. Results There were no differences in fibrinogen level between patients and controls. The prevalence of IgG anti-EB with MIF was 78% in patients and 24% in control subjects (P =.0001; odds ratio [OR], 11.3; 95% confidence interval [CI], 4.7-27.2). Prevalence of IgG anti-EB with ELISA was 75% in patients and 16% in control subjects (P = .0001; OR, 15.7; 95% CI, 6.1-40). There were no differences in IgA anti-EB titers. Bacterial DNA was detected in 67% of atheromatous plaques versus 12% of pudendal arteries (P = .0001) and 4% of saphenous veins. A weak correlation was found between seropositivity and the presence of intravascular DNA. Conclusions Our results support the hypothesis that C pneumoniae is related to the pathogenesis of atherosclerotic peripheral artery occlusive disease.