Efficient and sensitive diagnostic methods are needed in the management of virus infections in the central nervous system. There is a demand for an evaluation of the sensitivity of PCR methods for early diagnosis of meningitis due to herpes simplex type 2 (HSV-2) and varicella-zoster virus (VZV). The objective of this study was to evaluate real-time PCR in the detection of HSV-2 and VZV DNA from cerebrospinal fluid (CSF) for etiological diagnoses in clinically well-characterized cases of primary and recurrent aseptic meningitis. Samples from 110 patients, 65 of whom were diagnosed with or were strongly suspected of having HSV-2 meningitis and 45 with acute aseptic meningitis of unknown causes, were analyzed. Results were compared with the outcome of nested PCR for HSV-2 infection. Clinical parameters were analyzed in relation to CSF viral load. With real-time PCR, HSV-2 DNA was found in CSF from 80% (52/65) of patients with clinical HSV-2 meningitis compared to 72% (47/65) found by nested PCR. The sensitivity of real-time HSV-2 PCR was found to be 87% (33/38) in primary and 70% (19/27) in recurrent meningitis. The HSV-2 viral load was significantly higher in primary than in recurrent meningitis and correlated with the degree of inflammation. VZV DNA was detected in 2 of 45 samples (4.4%). Real-time PCR for the diagnosis of HSV-2 meningitis was evaluated in a large, clinically well-characterized sample of material and found to identify more cases than nested PCR in the group of patients with recurrent meningitis. Quantification of DNA enables further research of disease prognosis and treatment.