Backgroud and Objectives: To assess the spontaneous clearance of untreated Chlamydia trachomatis infections and factors correlated with the process. Study Design: Spontaneous clearance was assessed through review of laboratory database, chart review, and laboratory testing using direct immunofluorescence (DFA) and polymerase chain reaction (PCR) tests on C. trachomatis culture transport media from patients with negative chlamydial cultures. Specimens (C. trachomatis culture transport media) were obtained from patients attending a Birmingham, Alabama sexually transmitted diseases clinic. The study group consisted of patients with positive cultures for C. trachomatis who had repeat specimens obtained for culture within 45 days of initial observation and who had not received recommended therapy for chlamydial infection in the interval between the two tests. Results: Of 74 evaluable patients, 24 (32%) had negative follow-up cultures. Culture transport media for these 24 culturenegative patients were tested with DFA or PCR assays for chlamydial infection, and 3 (13%) were positive. Culture positivity rates declined significantly with increasing age and duration of follow-up. Interval treatment with benzathine penicillin resulted in apparent resolution of infection in 9 of 10 patients. Neither a history of a C. trachomatis-associated syndrome nor treatment with cefixime, metronidazole, or antifungal agents was associated with clearance of infection. Conclusions: These results are consistent with host responsemediated resolution of infection in a minority of patients and have implications regarding public health efforts to control chlamydial infection.