Herpes simplex virus (HSV) is prevalent worldwide and known as a notorious opportunistic pathogen ofimmunocompromised patients. During the course of HSV treatment, acyclovir (ACV)-resistant HSV strains may emerge, causing many clinical complications. Because few studies in this area showing the presence and/or frequency ofACV-resistant HSV are available, the authors evaluated the sensitivity of HSV isolated from 3 hematopoietic stem cell transplant recipients and 24 normal patients observed in a University Hospital in Rio de Janeiro, Brazil. Twenty-seven HSV isolated in VERO cells and typed by the direct immunofluorescence assay were tested for their susceptibility to various concentrations ofACV by the dye-uptake method. The susceptibility of the HSV strains was expressed as ED50 (the concentration of drug reducing viral cytophatic effect by 50%). The sensitivity to ACV by the dye-uptake assay revealed that 25 samples (92.6%) were sensitive to ACV concentrations < 1.5 microg/mL. One sample (3.7%) showed intermediate susceptibility (1.56 microg/mL) and one other sample (3.7%) showed resistance to ACV concentrations above the cut-off (2 microg/mL). The presence of resistance in immunocompetent patients could be the result of the frequent use of ACV for the treatment of recurrence. The routine use of HSV susceptibility testing is fundamental in the clinical suspicion of resistance not only for the knowledge of the incidence of HSV resistance in Brazil, but also to understand the mechanism of HSV resistance.