As part of a European multicenter study designed to determine the optimal combination and order of a mixed-modality vaccine against acquired immunodeficiency syndrome, rhesus monkeys received a combination of three different vectors, all expressing the same Simian Immunodeficiency Virus (SIV) genes followed by mucosal challenge with highly pathogenic SIV. In the study reported here, animals were primed with DNA followed by one booster immunization with Semliki Forest Virus (SFV) and two immunizations with modified Vaccinia Ankara (MVA). To address the relevance of mucosal immunization, we compared systemic versus a combination of systemic and mucosal antigen application. Although all vaccinees became infected after intrarectal challenge with SIV, most (six of eight) were protected from profound loss of CD4+ cells. In addition, vaccinees showed lower viral loads than did controls (p < 0.05). Overall, these protective effects were more pronounced in those animals whose schedule included immunization via the mucosa. In summary, the vaccine regimen used here achieved one important criterion of efficacy: the suppression of disease development as indicated by conservation of CD4+ cells.