The presence of nucleotide sequences specific for each of herpes simplex virus (HSV) serotypes was demonstrated. These sequences were applied for dot DNA-DNA hybridization and for PCR for rapid DNA diagnosis of HSV infections. These sequences were found by molecular cloning of HSV-DNA fragments after digestion of DNA by KpnI enzyme. The type 1-specific sequence was found around the 5' end of BamHI B-fragment in the L region of type 1 DNA (corresponds to alpha gene 27, promotor-regulatory region) and the type 2-specific sequence was around the junction region of the L and S of type 2 DNA (corresponds to a' sequence). Both simple dot blot hybridization and PCR of HSV DNA's, employing these type-specific nucleotide sequences, were proven to be much more useful than immunofluorescence in terms of type-specific diagnosis of HSV infections.