Integration of exogenous DNA into modified vaccinia Ankara (MVA) is often accomplished using mapped deletion sites in the viral genome. Since MVA has a large capacity (> or =30kb) for foreign gene inserts and a limited number of unique integration sites, development of additional integration sites is needed to take full advantage of the extraordinary capacity for foreign gene insertion. In this report, we evaluate an alternative insertion site known as intergenic region 3 (IGR3). Recombinant MVA carrying the cytomegalovirus pp65 gene in IGR3 (rMVA-pp65-IGR3) demonstrated expression and genetic stability of the insert gene upon passage. Immunization of transgenic HLA-A2 mice with rMVA-pp65-IGR3 induced robust antigen-specific immune responses. Moreover, rMVA-pp65-IGR3-infected human EBV-transformed B cell lines were able to stimulate high levels of pp65-specific memory T cell responses in human PBMCs. These data support the usage of IGR3 for the development of highly immunogenic rMVA vaccines for clinical or veterinary use.