The widespread use of acyclovir (ACV) could increase the prevalence of herpes simplex virus (HSV) ACV-resistant isolates, and a screening assay are thus important for routine surveillance of the ACV susceptibility of HSV. A screening dye-uptake assay was developed, based on the conventional dye-uptake assay [J. Biol. Stand. 14 (1986) 201]. The susceptibility of HSV was measured by testing two virus dilutions (10(-1) and 10(-2)) against two ACV concentrations (5 and 10 microM) on Vero cells and expressed as a reduced percentage of viral replication. The reproducibility was evaluated with HSV1 and HSV2 ACV-sensitive and ACV-resistant reference strains introduced as controls in successive series. The dye-uptake by Vero cells, the growth capacity of the HSV strains and the reduction of the viral replication in the presence of acyclovir varied by less than 14, 20 and 30%, respectively. This assay allowed the detection of a heterogenous population containing as few as 20% of ACV-resistant strain. The screening test was applied to 500 HSV isolates in a prospective study, and over 95% of the HSV isolates tested were characterised using a single test. This test appeared to be half the cost and much easier to carry out than the conventional dye-uptake assay, and consequently is well suited for large scale surveillance.