The acyclic nucleotide analog (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) is a potent and selective inhibitor of herpesviruses. Cells preincubated with HPMPC are refractory to herpes simplex virus type 2 (HSV-2) infection for several days after removal of the drug from the medium. A single administration of 30 mg of HPMPC per kg of body weight 4 days prior to virus infection intraperitoneally with HSV-2 (strain G) completely protected mice from death, and the protective effect was dose dependent. HPMPC was equally efficacious in protecting mice when the same total amount of the drug was administered as a single dose as when it was given daily in several smaller doses (5 mg/kg with treatment initiation at 3 h postinfection [p.i.], 90 versus 80% survival, respectively). In contrast, ganciclovir [9(1,3-dihydroxy-2-propoxymethyl)guanine] was more efficacious when it was given daily than it was when it was given less than daily in a late stage of HSV-2 infection (100 mg/kg; when mice were treated 96 h p.i., 80 versus 50% survival, respectively; when mice were treated 120 h p.i., 60 versus 20% survival, respectively). Therefore, single doses of HPMPC were more effective than ganciclovir in protecting mice from death (80 versus 20% survival, respectively; P less than 0.05), whereas there was no difference when the drugs were given daily (50 versus 60% survival, respectively). Our studies suggest a potential of HPMPC for conventional and prophylactic treatments of herpesvirus infections with infrequent drug administration.