The new HERPCHEK (Dupont, No. Billerica, MA) enzyme immunosorbent assay (EIA) was used in a double-blind clinical study for rapid and specific detection of ocular herpes simplex virus (HSV) infection. This 4-hour assay can be used to demonstrate conclusively the presence of HSV antigen without culture and thereby rapidly differentiate between HSV and other clinically similar ocular infectious diseases. Ocular samples were collected from 180 individuals including 30 patients with acute HSV, 90 with latent HSV (ie, currently asymptomatic but with a positive history), 11 with acute or latent varicella zoster virus, 30 with nonherpetic infections (due to adenovirus, Acanthamoeba or bacteria), and 19 normal controls. A clinical diagnosis was made by one of us (DPL) and duplicate tear-film samples obtained by swabbing the conjunctival cul-de-sac and cornea. Coded samples were tested by routine viral culture on Vero cell monolayers and also were run independently in the HERPCHEK test. During active HSV infection, the HERPCHEK correlated 100% with clinical diagnosis, and virus culture correlated 90% with clinical diagnosis. In all latent HSV ocular infections, other nonherpetic ocular infections and normal samples, both the HERPCHEK and culture assays were negative.