Background: Sexually transmitted diseases are often caused by one or more microorganisms, and asymptomatic carriage and transmission may be of significance. Testing for more than one organism in a single assay could be a useful approach to laboratory diagnosis. Methods and Results: A multiplex polymerase chain reaction (PCR) assay was developed that employed specific primers targeted to the 7.5-kb cryptic plasmid of Chlamydia trachomatis, the cppB gene of the 4.2-kb cryptic plasmid of Neisseria gonorrhoeae, the 140-kd major adhesion protein gene of Mycoplasma genitlium, and the urease gene of Ureaplasma urealyticum. All four polymerase chain reaction products were detectable by agarose gel electorphoresis and were confirmed by Southern hybridization using fluorescein isothiocyanate-labeled oligonucleotide probes and enhanced chemiluminescent detection. Using purified DNA preparations, multiplex PCR had a reproducible detection limit of 1 fg of C. trachomatis DNA, 100 fg of N. gonorrhoeae DNA, and 10 fg U. urealyticum DNA and M. genitalium DNA, which converts to 1-2 genomic equivalents (ge) of C. trachomatis and N. gonorrhoeae, 4 ge of M. genitalium, and 10 ge U. urealyticum. Multiplex PCR was compared with individual uniplex polymerase chian reaction PCR assays by testing 117 first-void urine samples (91 men, 26 women) from Canadian or Kenyan patients. Multiplex PCR detected 45 of 46 (97.8%) urines with C. trachomatis DNA, 42 of 42 (100%) urines with N. gonorrhoeae DNA, 17 of 17 (100%) urines with U. urealyticum DNA, 4 of 4 (100%) urines with M. genitalium DNA, 12 of 12 urines that had DNA from two bacteria, and 2 of 2 urines with DNA from three bacteria. Multiplex PCR correctly identified bacteria in 92 of 93 urines for an overall sensitivity of 98.9%. Specificity calculations were 100% for C. trachomatis (71/71), N. gonorhoeae (75/75), U. urealyticum (100/100), and M. genitalium (113/113). Conclusions: Multiplex PCR provided a single sensitive and specific test for the detection of four bacteria in first-void urine samples. Testing of first-void urine samples by multiplex PCR could facilitate studies aimed at improving our understanding of the epidemiology of these important sexually transmitted diseases.