Herpes simplex virus (HSV) causes several clinical manifestations in both normal and immunocompromised hosts; this agent is the most frequently detected virus in diagnostic laboratories. Recovery of the virus in cell culture is considered the "gold standard" for detection of this virus from sources other than cerebrospinal fluid. LightCycler is a newly developed, commercially available system designed to rapidly perform PCR, with real-time detection of PCR products by a fluorescence resonance energy transfer assay. We compared the detection of HSV for 200 specimens (number of genital specimens, 160; number of dermal specimens, 38; number of ocular specimens, 2) by shell vial cell cultures (MRC-5) and by LightCycler PCR. Of a total of 88 (44%) HSV strains detected, 69 (78%) were detected by both shell vial cell cultures and LightCycler PCR (DNA polymerase target). A total of 19 (22%) specimens were detected exclusively by LightCycler PCR. No specimens were positive by the shell vial assay only. All 19 discrepant samples had HSV DNA detected by an independent PCR directed to the thymidine kinase gene of the virus. The melting curve analysis feature of the LightCycler instrument identified identical genotype results for HSV type 1 (HSV-1) and HSV-2 from 84 of 88 (96%) positive samples. Specimens can be extracted, target HSV DNA can be amplified, and HSV PCR products can be identified by genotype within 2 h after receipt of specimen into the laboratory. The increased level of accurate identification (all 88 positive samples) compared with that of shell vial cell culture (69 of 88 samples identified as positive) and the agreement of LightCycler PCR results with all shell vial positive results indicate the potential for routine implementation of this technology for laboratory diagnosis of HSV infections.