Important progress in the diagnosis of Chlamydia trachomatis (C. trachomatis) includes the development of nucleic acid amplification techniques such as polymerase chain reaction (PCR) and ligase chain reaction (LCR). Commercial kits are available, but they are costly, sporadic in availability, must be imported, and are economically beyond the reach of common people. To overcome this limitation, most research laboratories have standardized their in-house-developed PCR methods for diagnosing this infection. However, each laboratory has to spend a great deal of time and money to accomplish this. Published reports do not always elaborate the steps involved in standardizing a test so that it can immediately be reproduced in another setting. In the present study we attempted to elaborate the steps involved in standardizing a sensitive and specific PCR technique followed by hybridization with specific C. trachomatis probe to diagnose this infection in cervical, introital, and urine specimens, and used it to determine the infection rate in a clinical population.