The polymerase chain reaction was used to detect major outer membrane protein (MOMP) gene sequences from the three species of Chlamydia. Using three primer pairs and one restriction enzyme digestion, three distinct genotypes, corresponding to the three species, Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci, were demonstrated. C. trachomatis was amplified by all three primer pairs and the amplified fragment was digested by EcoRI. C. pneumoniae was amplified by only two of the three primer pairs, and the amplified fragment was digested by EcoRI. C. psittaci was amplified by only two of the pairs and the amplified fragment was EcoRI-resistant. C. trachomatis was detected in direct patient specimens, tissue culture specimens, and fixed specimens, and all serovars of C. trachomatis were detectable. The polymerase chain reaction can detect and differentiate the three species of Chlamydia and may prove a valuable diagnostic tool.