Pooling urogenital specimens for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by nucleic acid amplification tests is an attractive alternative to individual testing. As pooling can reduce the costs of testing as well as labor, it has been advocated for use in resource-poor settings. However, it has neither been widely adopted nor evaluated for use in developing countries. We evaluated the practical use of pooling first-catch urine (FCU) specimens for the detection of C. trachomatis and N. gonorrhoeae from 690 men in Mumbai, India, by PCR. FCU, urethral smears, and swabs were collected from men seen at two sexually transmitted infection (STI) clinics. All laboratory testing was done at the Lokmanya Tilak General Hospital. Gram stain smears and culture isolation for N. gonorrhoeae were performed. Each FCU was tested individually and in pools using the Roche Amplicor PCR for C. trachomatis and N. gonorrhoeae with an internal control for inhibition. Specimen pools consisted of aliquots from five consecutively processed FCUs combined into an amplification tube. An optical density reading of > or =0.20 indicated a pool for which subsequent testing of individual samples was required. Prevalence by PCR on single specimens was 2.2% (15/690) for C. trachomatis and 5.4% (37/690) for N. gonorrhoeae. Compared to individual FCU results, pooling for C. trachomatis and N. gonorrhoeae had an overall sensitivity of 96.1% (50/52). Specificity was 96.5% (83/86) in that three pools required single testing that failed to identify a positive specimen. Pooling missed two positive specimens, decreased the inhibition rate, and saved 50.3% of reagent costs. In this resource-limited setting, the use of pooling to detect C. trachomatis and N. gonorrhoeae by PCR proved to be a simple, accurate, and cost-effective procedure compared to individual testing.