The development of techniques for the amplification of DNA and RNA opened the way for the creation of extremely specific, sensitive, and rapid diagnostic tests for the detection of viral infections of the central nervous system. Polymerase chain reaction (PCR) and reverse transcription PCR diagnostic assays have revolutionized the approach to the diagnosis of important viral pathogens-in particular, enteroviruses (EVs), herpes viruses, and JC virus (JCV). These molecular approaches to diagnosis have led to improvements in clinical outcome and patient care. Additionally, their use has permitted a better understanding of the natural history and clinical spectrum of the syndromes caused by these important human pathogens. This article summarizes the current understanding with regard to the available, molecularly based, diagnostic assays for the detection of EVs, herpes viruses, and JCV.