As the single-most potent virulence factor of the vaccinia virus, the 13.8-kDa protein enhances viral replication in the brain by an unknown mechanism. Due to the high energy demands of the brain and the at times inadequate energy supply and small energy reserves to support physiologic activity, the ability of this organ to support energy requirements for replication of a virus is unlikely. We investigated the possible role of the 13.8-kDa protein in the enhancement of adenosine triphosphate (ATP) utilization in the brain to sustain viral replication. In vitro and in vivo monitoring and comparison of ATP levels in mouse brain tissue infected with a wild-type vaccinia virus or a 13.8-kDa deletion strain (vGK5) revealed differences in ATP utilization and a significant difference in ATP levels in vivo after 5 days of infection. Because of poor replication of the wild-type Lister vaccinia virus in the brain, a role for the 13.8-kDa protein in the modulation of ATP levels to support viral replication in the brain could not be conclusively implicated. Evaluation of the amino acid sequence and predicted secondary structure of the 13.8-kDa protein and sequence and structural homologs thereof provided evidence of putative dimerization and adenine binding sites and a possible kinase-related function for this protein.