Varicella-zoster virus (VZV) infection can be definitively diagnosed by isolation of virus in cell culture, a process that usually takes 7-14 days. In order to facilitate the more rapid detection of this virus, we developed a technique for hybridization of DNA from clinical specimens using an in vitro-labeled mixture of cloned fragments of VZV DNA as a probe. The assay can be completed in 36-48 hr and can be successfully carried out in the range of 10 pg to 10 ng of viral DNA. In analyses of 38 specimens from patients with a clinical diagnosis of VZV infection, the results of viral isolation and this assay were highly concordant. The sensitivity of standard cell culture for detection of VZV was 58%, whereas the sensitivity of the assay was 76%, not significantly different (P = 0.14). The specificity of cell culture was 100%, whereas that of the assay was 94% (P = 0.49). The technique appears to be sensitive, specific, and useful for analyses of tissues and body fluids.