Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency, and speed under isothermal conditions. To evaluate the usefulness of LAMP for diagnosing central nervous system infection with herpes simplex virus (HSV), we compared the LAMP method with real-time PCR, using samples that were previously tested by nested PCR. We examined 69 cerebrospinal fluid (CSF) samples from patients suspected of having HSV infection of the central nervous system. The results of the real-time PCR analysis and nested PCR assay were in complete accord. When nested PCR was regarded as standard, the sensitivity of LAMP was 81%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 90%. Although further improvement is necessary for the wide spread use, the LAMP method might be applicable to diagnosis of HSV infection of the central nervous system.