BACKGROUND: Herpes simplex virus (HSV) is associated with a insignificant skin lesion, keratitis, encephalitis, congenital infection, sexually transmitted disease, or cervix cancer. There are two types of serogroup, HSV-1 and HSV-2. HSV-1 makes the lesion mainly on the above-waist area and HSV-2 makes the lesion mainly on the below-waist area. To diagnose the HSV infection, immunological or cultural methods usually have been used until now. But they are not satisfactory in terms of sensitivity, specificity, and ease of application. Recently the polymerase chain reaction (PCR) was developed. Because of the exponential nature of the amplification, this method can detect extremely small amount of DNA. We compared nested PCR with cultural method for HSV detection. METHODS: We obtained 61 specimens from the lesions of oral mucosa, face, and genital area. Samples were inoculated into the monolayer from the African green monkey kidney cell(Vero). When the slide showed cytopathic effect(CPE), HSV infection was confirmed, After extracting DNA from 61 samples, we amplified HSV DNA using nested PCR with the primers against the gene encoding glycoprotein (gD) of HSV-1 and HSV-2. RESULTS: We found 632 bp band after the 1st PCR round and 271 bp band after the 2nd PCR round with HSV-1 specific primers. HSV-2 revealed 428 bp band after the 1st PCR round and 231 bp band after the 2nd PCR round. Nested PCR showed analytical sensitivity at 10(-9) g of DNA in HSV-1 and 10(-10) g of DNA in HSV-2. Viral culture was positive in 36%, nested PCR detected HSV DNA sequence in 54% of samples. Nested PCR typed HSV, HSV-1 in 67%, HSV-2 in 39%, and mixed type in 6% of PCR-positive samples. All isolates from above-waist area were HSV-1. Seventy seven percent of 13 isolates from below-waist area were HSV-2 and 38% were HSV-1. CONCLUSIONS: Nested PCR offers a rapid, simple, and sensitive test for HSV infections of skin and mucosa.