Viral isolation is the standard method for the detection of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in clinical specimens. This study describes the development of a type-specific polymerase chain reaction (PCR) assay for detection and typing of HSV-1 and HSV-2, and a comparison of its sensitivity with that of isolation in a clinical setting. Specimens from patients presenting with genital ulcers were tested for the presence of HSV by both methods. Oligonucleotide primers were selected to enable type-specific amplification of HSV-1 and HSV-2 DNA. Conditions were optimized to allow detection and typing from a single reaction tube using a multi-primer PCR method. When compared with PCR, the sensitivity of isolation was 67% and the specificity 97%. This protocol allowed rapid, sensitive and accurate detection and typing of HSV with a single PCR assay.