To shorten the time and improve the accuracy of diagnosis of vaginal trichomoniasis, a novel, one-tube, nested PCR, which targets a family of 650-bp specific DNA repeats from the Trichomonas vaginalis genome, has been developed. Samples were prepared by a rapid boiling method and the PCR products analysed by gel electrophoresis. A 290-bp DNA fragment was observed in all positive cases. No cross-reaction with any other pathogens, including the Pentatrichomonas hominis and Giardia lamblia used as controls, was found. Using the assay, one genome-equivalent of T. vaginalis in 20 microliters vaginal discharge can be detected and diagnosis can be made within 6 h. When 165 clinical specimens were examined by wet amount, culture and the PCR assay, 16 were found positive for T. vaginalis by both culture and PCR, whereas only nine of these 16 cases were found to be positive by examination of wet mounts. No PCR-negative cases were positive by wet mount or culture. This new assay appears to be a simple, rapid, accurate and sensitive method for the diagnosis of vaginal trichomoniasis.