It has been reported previously that some thiosemicarbazone compounds have prophylactic activity against smallpox disease and therapeutic activity against vaccinia virus (VV) infections. In these studies, isatin-beta-thiosemicarbazone (IBT) and marboran were administered once daily by intraperitoneal (ip) injection to mice using 30, 10 or 3 mg/kg for 5 days beginning 24, 48 or 72 h after inoculation with VV or cowpox virus (CV). Both compounds were highly effective (p < 0.01) at preventing mortality due to VV even when treatment was delayed up to 72 h postinfection. In CV-infected mice, neither IBT nor Marboran were effective in preventing mortality at any dosage tested when administered at 24 h postinoculation. Viral replication in liver, spleen and kidney was delayed or reduced by 100-to 10,000-fold by 10 mg/kg of marboran, but not IBT, in VV infections. Neither compound was effective against CV infection. Neither IBT nor marboran treatment of mice cutaneously infected with VV or CV reduced viral replication or clinical disease. These results suggest that this class of compound has little therapeutic potential for orthopoxvirus infections since the in vivo activity against CV, a surrogate virus for variola, is lacking.