An assay to detect herpes simplex virus (HSV) DNA in clinical specimens has been developed. It utilizes nucleic acid hybridization with a 32P-labeled DNA probe prepared from a fragment of HSV DNA cloned in a plasmid vector. This assay can detect 5 X 10(4) plaque-forming units of cell-free HSV and as few as four virus-infected cells. The assay has a sensitivity of 78% and a specificity of 100% compared to virus culture for the detection of HSV in swab specimens from genital lesions. No hybridization is observed with uninfected, varicella-zoster virus infected, or cytomegalovirus infected cells, and specimens from herpes zoster lesions are uniformly negative. While hybridization with a 32P-labeled probe is not optimally suited for routine diagnostic use, this report establishes the feasibility of using nucleic acid hybridization to detect HSV in clinical specimens.