Diagnosis of central nervous system (CNS) infection with herpes simplex virus (HSV) requires sensitive and rapid techniques. PCR therefore is considered to be the diagnostic gold standard in these cases. However, current PCR protocols are time-consuming and labor-intensive. In addition, the need for post-amplification manipulations increases the risk of laboratory contaminations with amplified products. In order to improve conventional PCR techniques we compared our current semiautomated HSV-PCR-ELISA assay with a new micro-volume rapid-cycle PCR system that combines real-time monitoring and fluorescence melting-curve analysis without the need for post-amplification sample manipulations. Spiking experiments with supernatants of tissue culture-grown HSV type 1 (HSV-1) and type 2 (HSV-2) in HSV-negative control cerebrospinal fluid (CSF) and sterile water revealed that the new rapid cycle PCR protocol is as sensitive and specific as the PCR-ELISA. Furthermore, a mismatch (G:T) within the probe-targeted region of the HSV-2 glycoprotein B gene decreases the probe/product melting temperature (Tm) from 69 degrees C for HSV-1 to 64 degrees C for HSV-2, enabling the simultaneous identification of the two HSV genotypes by melting-curve analysis within one run. This type specificity of the system was confirmed with 30 genital swabs previously analyzed for the presence of HSV-1/2 in cell culture. While our current PCR-ELISA method needs up to 1 day from sample preparation to result generation, the new procedure takes only 1 h. We consider this system as a promising new tool for the analysis of HSV DNA in CSF and in other human body fluids as well as for the diagnosis of other infectious agents where rapid diagnosis, high sensitivity and specificity are required.