Specimens submitted for diagnosis of herpes simplex virus (HSV) infection were inoculated into shell vials and reacted with a commercial DNA probe kit (Pathogene; Enzo Biochem, Inc., New York, N.Y.) and an immunofluorescence assay at 16 h postinoculation. The results were compared with isolation of the virus in conventional tube cell cultures. Of 504 specimens, 105 (20.8%) were positive for HSV. Of the 105, 93 HSV-positive specimens (89%) were detected by all three assay systems. Maximum detection of HSV (100 of 105 [95%]) was obtained by probe or monoclonal antibody assay in shell vials, which had sensitivities of 98 and 97%, respectively, compared with viral recovery in conventional tube cell cultures (mean time for recognition of cytopathic effects, 2 days). Both shell vial assays were 99% specific. The DNA probe kit may be used as an alternative to a monoclonal antibody and fluorescence assay in shell vials as a diagnostic method for rapid laboratory detection of HSV infection.