Background: Secondary infertility in developing countries is mostly attributable to blockage of the fallopian tubes due to adhesions caused by reproductive tract infections. There is a dearth of information on the prevalence and causes of secondary infertility from Pakistan. This paper presents results on factors associated with secondary infertility among married women in Karachi, Pakistan. Methods: A matched case-control study was conducted. Cases were women aged 15-35 years with history of at least one previous conception and currently seeking treatment for secondary infertility. Controls were women residing in the neighborhood of cases with at least one live birth and not taking treatment for secondary infertility. The age of controls was matched by ±5 years to that of cases. Data was collected from June to August 2003. Conditional logistic regression was used to determine crude and adjusted odds ratios (OR) with corresponding 95% confidence intervals (CI) for factors associated with secondary infertility. Results: The final multivariate logistic regression model revealed that after adjusting for age, cases were more likely to be the housewives (AOR = 2.6, 95% CI:1.5-4.4), had used inappropriate material to absorb blood during menstruation (AOR = 9.0, 95% CI: 5.0-16.4), and at their last delivery, had a birth attendant who did not wash hands with soap and water (AOR = 3.0, 95% CI: 1.4-5.7). Moreover, women with secondary infertility were more likely to report current or past history of having STI symptoms (AOR = 3.6, 95% CI: 2.4-5.6) and use of intra-vaginal indigenous medicines during their last post-partum period (AOR = 3.1, 95% CI: 1.6-5.7). Conclusion: We recommend health education and awareness messages for safe practices during menstruation, delivery, and the postpartum period for women in general. Additionally, sanitary napkins should be made available at an affordable cost, and safe delivery kits should contain educational/pictorial brochures for appropriate hand washing skills.