Vaccinia virus (VACV) is the vaccine that was used to eradicate smallpox and is being developed as a recombinant vaccine for other pathogens. Removal of genes encoding immunomodulatory proteins expressed by VACV may enhance virus immunogenicity and improve its potential as a vaccine. Protein A41 is a candidate for removal, having sequence similarity to the VACV chemokine-binding protein, vCKBP, and an association with reduced inflammation during dermal infection. Here, it is shown that, at low doses, VACV strain Western Reserve (WR) lacking A41L (vDeltaA41L) was slightly more virulent than wild-type and revertant controls after intranasal infection of BALB/c mice. The primary immune response to vDeltaA41L was marked by an increase in the percentage of VACV-specific gamma interferon-producing CD8(+) T cells and enhancement of cytotoxic T-cell responses in the spleen. However, this augmentation of cellular response was not seen in lung infiltrates. Splenic CD8(+) T-cell responses were also enhanced when VACV strain modified vaccinia virus Ankara (MVA) lacking A41L was used to immunize mice. Lastly, immunization with VACV MVA lacking A41L provided better protection than control viruses to subsequent challenge with a 300 LD(50) dose of VACV WR. This study provides insight into the immunomodulatory role of A41 and suggests that MVA lacking A41 may represent a more efficacious vaccine.