We have previously demonstrated that peritoneal macrophages (M phi S) from C3H mice were resistant to in vitro infection by ectromelia virus, following activation by intraperitoneal injection of the immunomodulator Corynebacterium parvum. In contrast, resident and mineral oil-elicited M phi S were fully susceptible to virus infection. This report analyzes the infectious cycle of ectromelia virus in C parvum-activated and mineral oil-elicited M phi S and demonstrates that an abortive infection occurred in the activated M phi S that blocked the infectious cycle prior to the release of DNA from the infecting virions. The kinetics of adsorption of radiolabeled virus were similar in both susceptible and resistant M phi cultures; however, viral-induced incorporation of uridine and thymidine occurred only in the mineral oil-elicited and not the C parvum-activated M phi S. In addition, the late protein hemagglutinin was only detected in infected cultures of susceptible mineral oil-elicited M phi S. An electron micrographic analysis of the infectious cycle indicated that the adsorption of virus to the plasma membrane, uptake into lysosomes, and the primary undercoating and release of viral cores into the M phi cytoplasm were identical in both M phi types. In contrast, secondary uncoating (release of genomic DNA from the viral cores into the cytoplasm) was never detected in infected C parvum M phi S. These data are consistent with our previous findings and with the hypothesis that activation of M phi S by C parvum induces an interferon-mediated resistance to ectromelia virus infection.