5,6-Dihydro-5-azathymidine (DHAdT), a nucleoside antibiotic inhibitory for herpes simplex virus (HSV) in cell cultures (H. E. Renis, Antimicrob. Agents Chemother. 13: 613-617, 1978), was evaluated in mice with experimental HSV infections. DHAdT protected mice infected with HSV type 1 (HSV-1) when the virus was inoculated intravenously and the drug was given by subcutaneous or oral routes. The activity observed was dependent on the dose and schedule of treatment. Doses of 100 to 400 mg/kg given three to four times daily (at 4-h intervals) for 4 to 5 days gave greater protection than less frequent treatment for shorter time intervals. DHAdT treatment reduced the rate of isolation as well as the HSV-1 titers in homogenates prepared from spinal cords and brains, whereas the titers in kidney homogenates were only marginally affected. The above treatment regime with DHAdT afforded only partial protection to mice infected intracerebrally with HSV-1 or mice inoculated intravaginally with HSV-1 or HSV-2. The antiviral activity of DHAdT was reversed by the co-administration of thymidine. Under these conditions, DHAdT was not toxic in mice.