The detection of herpes simplex virus (HSV) antigen by means of an enzyme amplified ELISA was investigated for rapid screening of acyclovir (ACV) resistance. Vero cell monolayers were inoculated in the presence of different concentrations of ACV. When cytopathic effect was present, the culture supernatants were tested by ELISA. The absorbance values were found to correlate with the results of virus yield and plaque reduction assays. The comparison between absorbance values obtained in the presence of 10 microM ACV and in the absence of drug provided the basis for a simplified sensitivity test. The use of a single ACV concentration allowed discrimination between ACV-resistant and ACV-sensitive reference strains, the detection of ACV-resistant virus mixed in the proportion of 10% with ACV-sensitive virus, and a study of the emergence of an ACV-resistant virus population in serial samples taken from experimental rabbit keratitis. The simplified susceptibility assay is a sensitive and convenient method for rapid screening of HSV resistance to ACV.