OBJECTIVE: We sought to estimate the rate of spontaneous resolution of asymptomatic Chlamydia trachomatis in pregnancy and to evaluate factors associated with its resolution. METHODS: A cohort of women enrolled in a large multicenter randomized bacterial vaginosis antibiotic trial (metronidazole versus placebo) that, when randomly allocated, had asymptomatic C trachomatis diagnosed by urine ligase chain reaction (from frozen archival specimens) between 160/7 and 236/7 weeks were included. The urine ligase chain reaction is a highly accurate predictor of genital tract chlamydial infection. A follow-up ligase chain reaction was performed between 24 0/7 and 296/7 weeks. RESULTS: A total of 1,953 women were enrolled in the original antibiotic trial; 1,547 (79%) had ligase chain reaction performed both at randomization and follow-up. Women receiving antibiotics effective against Chlamydia between randomization and follow-up or having symptomatic Chlamydia infection were excluded (26 women). Of the 140 women (9%) who were diagnosed as positive via the initial ligase chain reaction assay, 61 (44%) had spontaneous resolution of Chlamydia by the follow-up ligase chain reaction assay. Factors associated with spontaneous resolution included older age (P = .02), more than 5 weeks from randomization to follow-up (P = .02), and a greater number of lifetime sexual partners (P = .02). Using a logistic regression model, maternal age and a greater-than-5-week follow-up interval remained significant; for every 5-year increase in maternal age, the odds of a positive result on the ligase chain reaction test at follow-up decreased by 40% (odds ratio 0.6; 95% confidence interval 0.4-0.9). Race, substance abuse, parity, and treatment with metronidazole were not associated with spontaneous resolution. Gram stain score and vaginal pH at randomization and follow-up also were not associated. CONCLUSION: The prevalence of asymptomatic C trachomatis in pregnancy was 9%; infection resolved spontaneously in almost half of these women. The association of older age and increasing time interval to spontaneous resolution of Chlamydia is consistent with a host immune-response mechanism. © 2005 by The American College of Obstetricians and Gynecologists.