Today, the polymerase chain reaction (PCR) analysis of the cerebrospinal fluid (CSF) is commonly used for the diagnosis of herpesvirus infections of the central nervous system (CNS). Due to the great number of herpesviruses and limited amount of CSF, PCR application offers limited throughput when viruses are screened individually. The purpose of this preliminary study was to create and optimize a multiplex PCR (M-PCR) method that enables the simultaneous screening of five viruses including Herpes simplex virus type 1 and type 2 (HSV-1, HSV 2), Varicella-zoster virus (VZV), Cytomegalovirus (CMV) and Epstein-Bar virus (EBV) in CSF samples. Pairs of CSF samples, which were found positive for HSV-1, HSV-2, VZV and EBV but not for CMV by virusspecific PCR reactions, were examined by M-PCR. In order to determine the presence of multiple viruses, CSF samples were mixed and the examination was repeated. At the end of the study, expected virus DNAs could be determined in each sample and multiple viruses could be determined in the mixed samples by M-PCR. Although this study shows that five herpesviruses can be easily detected via a single amplification reaction, the method should be further evaluated by numerous clinical samples.