Vaginal trichomonosis is a highly prevalent infection which has been associated with human immunodeficiency virus acquisition and preterm birth. Culture is the current "gold standard" for diagnosis. As urine-based testing using DNA amplification techniques becomes more widely used for other sexually transmitted diseases (STDs) such as gonorrhea and chlamydia, a similar technique for trichomonosis would be highly desirable. Women attending an STD clinic for a new complaint were screened for Trichomonas vaginalis by wet-preparation (wet-prep) microscopy and culture and for the presence of T. vaginalis DNA by specific PCR of vaginal and urine specimens. The presence of trichomonosis was defined as the detection of T. vaginalis by direct microscopy and/or culture from either vaginal samples or urine. The overall prevalence of trichomonosis in the population was 28% (53 of 190). The sensitivity and specificity of PCR using vaginal samples were 89 and 97%, respectively. Seventy-four percent (38 of 51) of women who had a vaginal wet prep or vaginal culture positive for trichomonads had microscopic and/or culture evidence of the organisms in the urine. Two women were positive for trichomonads by wet prep or culture only in the urine. The sensitivity and specificity of PCR using urine specimens were 64 and 100%, respectively. These results indicate that the exclusive use of urine-based detection of T. vaginalis is not appropriate in women. PCR-based detection of T. vaginalis using vaginal specimens may provide an alternative to culture.