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Chlamydia pneumoniae (Cpn) has been proposed as a possible etiologic agent for multiple sclerosis (MS), but results of previous studies are conflicting.Using a nested case-control design, we examined the association between Cpn infection and MS in the Nurses’ Health Study (NHS) and Nurses’ Health Study II (NHS II) cohorts. Among 32,826 women in the NHS and 29,722 women in the NHS II with blood samples, 141 incident cases of definite or probable MS were documented. Each case was matched to two healthy controls on year of birth and NHS cohort. Serum samples were tested for the presence of Cpn-specific immunoglobin G antibodies using microimmunofluorescence.Cpn immunoglobin G seropositivity was positively associated with risk of MS (odds ratio [OR] = 1.7; 95% confidence interval [CI] = 1.1–2.7). This association did not change after adjusting for age at blood collection, ancestry, latitude of residence at birth, and smoking (OR = 1.9; CI = 1.1–3.1). Seropositivity for Cpn was only moderately associated with risk of relapsing-remitting MS (OR = 1.7; CI = 0.9–3.2), but it was strongly associated with risk of progressive MS (OR = 7.3; CI = 1.4–37.2). Geometric mean titers of Cpn-specific immunoglobin G antibody were similar in women with relapsing-remitting MS as compared with matched controls (44 vs 39), but they were elevated in women with progressive MS (99 vs 40).These results support a positive association between Cpn infection and progressive MS.