We developed a multiplex real-time PCR assay to simultaneously detect herpes simplex virus-1 (HSV-1) and HSV-2 genomes. TaqMan PCR primer pairs and fluologenic probes targeting the HSV-1 or HSV-2 gpD region were originally designed. The detection limit was 15 copies/tube, and the quantitative range was from 15 to 1.5 x 10(6) copies/tube of HSV-1 or HSV-2 DNA. The sensitivity of this assay was 10(3) times higher than the conventional PCR assay. No other herpes virus DNA-Cytomegalovirus, Epstein-Barr virus, human herpes virus 6, human herpes virus 7, or varicella-zoster virus-were detected by this assay. These results indicated that the multiplex real-time PCR assay is useful for rapidly diagnosing HSV-1 and HSV-2.