Three acyclic guanosine analogs with similar structures, the (R) and (S) forms of 9-(3,4-dihydroxybutyl)guanine and 9-(4-hydroxybutyl)guanine, were compared for antiherpes activity in vivo and in vitro. The three guanosine analogs were viral thymidine kinase-dependent inhibitors of virus multiplication. In cell cultures, (S)-9-(3,4-dihydroxybutyl)guanine was the least active of these three drugs against a variety of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) strains. This was also the case for a certain HSV-1 or HSV-2 strain in different cell lines. In cell cultures, (R)-9-(3,4-dihydroxybutyl)guanine and 9-(4-hydroxybutyl)guanine had similar antiherpes activities. However, in vivo in cutaneous HSV-1 infections in guinea pigs treated topically and in systemic HSV-2 infections in mice treated orally or intraperitoneally, only (R)-9-(3,4-dihydroxybutyl)guanine had a therapeutic effect. The extremely short half-life in plasma and the high clearance of 9-(4-hydroxybutyl)guanine as compared with those of (R)-9-(3,4-dihydroxybutyl)guanine probably made 9-(4-hydroxybutyl)guanine inefficacious when given intraperitoneally or orally to mice infected with herpesvirus. On the other hand, no kinetic differences between (R)-9-(3,4-dihydroxybutyl)guanine and 9-(4-hydroxybutyl)guanine were observed in penetration through guinea pig skin ex vivo, and no preferential metabolism of 9-(4-hydroxybutyl)guanine in skin was noted. We deduced that high thymidine levels in guinea pig skin preferentially antagonize the antiviral effect of 9-(4-hydroxybutyl) guanine in cutaneous HSV-1 infections.