The vaccinia virus (VV) genome encodes many proteins that are non-essential for virus replication in vitro but are involved in interactions with the host. The VV genes A46R and A52R were investigated as potential novel immunomodulatory factors. A46R and A52R encode intracellular proteins with amino acid similarity to signalling molecules and expression of these genes in mammalian cells interfered with IL-1 and Toll-like receptor signal transduction. To characterise both proteins in the context of virus infection and investigate their functions in vitro and in vivo, mutant viruses lacking either or both genes were constructed by transient dominant selection. Deletion of either or both genes did not affect growth properties in vitro. Both proteins are expressed early and late during virus infection. A46R seems to be predominantly cytoplasmic, whereas A52R seems to be predominantly membrane associated. Roles for A46R in blocking IL-1-mediated ERK activation and for A52R in potentiating IL-1-induced JNK activation are proposed. In an intradermal mouse model, infection with vDA46R produced a significantly larger lesion size when compared to the size of the lesions caused by infection with the control viruses. The mechanisms leading to this phenotype are unknown. In an intranasal mouse model, infection with vDA46R, vDA52R or vDA46RDA52R (vDD) results in attenuated phenotypes. The causes of such attenuations are likely to be related to different kinetics of cell recruitment to the infected lungs compared to the control viruses. In conclusion, both A46R and A52R contribute to virulence in a murine intranasal model of infection and A46R contributes to virulence in an intradermal model of infection.