A one-tube nested polymerase chain reaction (PCR) using primers derived from the major outer membrane protein gene sequence of Chlamydia trachomatis was compared with McCoy cell culture for the detection of the micro-organism in clinical specimens. Of 110 urogenital and ocular specimens, 21 were positive by culture and 25 by nested PCR; all 21 culture-positive specimens were also positive by nested PCR. After resolution of discrepant specimens as true-positive, sensitivity was 100% for nested PCR and 84% for cell culture, while specificity was 100% for both tests. One-tube nested PCR appears to be a rapid, highly sensitive and specific technique for diagnosing C. trachomatis infections.