To assess the role of Chlamydia in secondary infertility in a prospective study. Forty women with secondary infertility and 30 healthy term pregnant women of similar age composition were studied for past and present Chlamydia trachomatis infection. Women attending the outpatient Department of Obstetrics and Gynaecology with complaint of secondary infertility were enrolled as patients in the study. Forty women with secondary infertility formed the study group, and 30 healthy women served as the controls. Chlamydia IgG was detected by ELISA; titers of 1:320 or more were considered positive. Endocervical swabs were collected for culture on cycloheximide-treated McCoy cell lines, and ELISA was used to detect Chlamydia antigen. Hysterosalpingography was performed to assess tubal patency. A difference was expected between the prevalence of C. trachomatis infection in the infertile study subjects and fertile control group. Immunoglobulin G antibodies were present in 22 (55%) women with secondary infertility, whereas positivity was seen among 2 (5.5%) controls. Tubal occlusion occurred in 16 (63.6%) cases positive for chlamydial antibody. Sensitivity of chlamydial IgG antibody as a diagnostic marker for infertility was 72.7%, and specificity was 44.4%. The majority of Chlamydia IgG antibody-positive cases, 17 (77.2%), were symptomatic. Unfavorable obstetric history was found in 16 (72.7%) cases. Active infection was found in 12 (30%) cases with one (3.3%) case of current infection occurring in the controls. Prevalence of past chlamydial infection is strongly statistically significant in women with secondary infertility. Current infection was also found statistically significantly in these women. Immunoglobulin G antibody detection is an effective and noninvasive tool for detection of Chlamydia and a more viable option than HSG in a developing country such as India. Screening of women with secondary infertility for C. trachomatis is strongly recommended to allow early therapeutic interventions.