The present study was conducted to assess the usefulness of adding the multiplex nested-polymerase chain reaction assay of cerebrospinal fluid samples to routine diagnostic testing for herpesvirus encephalitis and to monitor the efficacy of therapy. Cerebrospinal fluid samples from 45 patients with presumed herpesvirus encephalitis were tested for herpes simplex virus, varicella-zoster virus, cytomegalovirus, human herpesvirus 6, and Epstein-Barr virus. Ten of the 45 patients were positive for a virus using the polymerase chain reaction assay: herpes simplex virus (n=5), Epstein-Barr virus (n=3), and herpes simplex virus plus the Epstein-Barr virus (n=2). Cerebrospinal fluid from two patients who had undergone acyclovir therapy gave negative results. Analysis of cerebrospinal fluid by multiplex polymerase chain reaction can be useful for establishing an accurate diagnosis and as a marker of the efficacy of therapy.