A series of 111 thiosemicarbazones of 2-acetylpyridine, 2-acetylquinoline, 1-acetylisoquinoline, and related compounds were evaluated as inhibitors of herpes simplex virus in vitro and in a cutaneous herpes guinea pig model. All derivatives tested were potent inhibitors of virus replication with mean 50% inhibitory concentrations of 1.1 micrograms/ml for both type 1 and 2 herpes simplex virus. Inhibitory concentrations for cellular protein and DNA synthesis were considerably higher for many compounds resulting in in vitro therapeutic indices ranging from greater than 100 (highly selective) to less than 1 (negatively selective). All compounds were tested for dermal toxicity following topical administration of saturated solutions in 1,3-butanediol to the shaved, depilated skin of guinea pigs. Approximately 50% of the compounds produced slight to no dermal toxicity whereas the remaining compounds produced moderate to severe dermal toxicity. 28 compounds were evaluated in the cutaneous herpes guinea pig model against herpes simplex virus type 1. A number of N4-monosubstituted 2-acetylpyridine thiosemicarbazones produced highly significant reductions in days to healing and lesion score without producing untoward dermal toxicity. Structure-activity relationships revealed that a reduction of the azomethine bond in the molecule (i.e., conversion of a thiosemicarbazone to a thiosemicarbazide) greatly diminished dermal toxicity apparently without producing a proportional decrease in antiviral activity.