DNA immunizations with glycoprotein 120 (gp120) of human immunodeficiency virus–1 (HIV-1) usually require boosting with protein or viral vaccines to achieve optimal efficacy. Here, we demonstrate for the first time that mice immunized with DNA encoding gp120 fused with proinflammatory chemoattractants of immature dendritic cells, such as β-defensin 2, monocyte chemoattractant protein–3 (MCP-3/CCL7) or macrophage-derived chemokine (MDC/CCL22), elicited anti-gp120 antibodies with high titers of virus-neutralizing activity. The immunogenicity was further augmented with the use of chemokine fusion constructs with gp140, gp120 linked to the extracellular domain of gp41 via a 14–amino acid spacer peptide sequence. This construct elicited antibodies with more effective neutralizing activity than corresponding constructs expressing gp120. Responses were dependent on physical linkage with chemokine moiety, as no immunity was detected following immunization of mice with DNA encoding a free mixture of chemokine and gp120. Although the route of immunization was inoculation into skin, both systemic and mucosal CD8⁺ cytolytic immune responses were elicited in mice immunized with DNA expressing MCP-3 or β-defensin 2 fusion constructs. In contrast, no cytotoxic T lymphocyte activity (CTL) was detected in mice immunized with DNA encoding gp120 either alone or as fusion with MDC. Therefore, the potential for broad application of this approach lies in the induction of mucosal CTL and neutralizing antibodies to HIV-1 envelope, both key requirements for prevention of viral transmission and clearance of pathogenic HIV from mucosal reservoirs.