Prompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and the possible initiation of antiviral therapy. In order to assist in arriving at a rapid diagnosis, real-time PCR assays have been developed for the detection of herpes virus DNA in patient specimens. A recently described set of real-time PCR assays using LightCycler technology enabled the parallel detection of DNA from herpes simplex virus by using a single LightCycler program. We set up HSV real-time PCR on the Lightcycler system using the Roche LightCycler HSV 1/2 Detection kit and evaluated this LightCycler assay in regard to the rapid detection and subtyping of herpes simplex virus (HSV) in the cerebrospinal fluid (CSF) of patients with herpes simplex infection of the central nervous system (CNS).　We also compared the results with those of the `in-house` nested PCR. The sensitivity of the LightCycler PCR assay was the same as that of the nested PCR assay. Furthermore, this system enabled the simultaneous identification of HSV-1/HSV-2　through the use of melting curve analysis. The total processing time for the detection and subtyping of HSV was less than 1 hour. Thus, LightCycler PCR has the advantages of rapid amplification and a reduced risk of contamination, and is a suitable method for diagnosis of  HSV infection in the CNS.