ABSTRACT To evaluate the immunogenicity of human immunodeficiency virus (HIV) type 1 p55 ^gag virus-like particles (VLPs) released by budding from yeast spheroplasts, we have analyzed the effects of yeast VLPs on monocyte-derived dendritic cells (DCs). Yeast VLPs were efficiently incorporated into DCs via both macropinocytosis and endocytosis mediated by mannose-recognizing receptors, but not the mannose receptor. The uptake of yeast VLPs induced DC maturation and enhanced cytokine production, notably, interleukin-12 p70. We showed that yeast membrane components may contribute to DC maturation partly through Toll-like receptor 2 signaling. Thus, Gag particles encapsulated by yeast membrane may have an advantage in stimulating Gag-specific immune responses. We found that yeast VLPs, but not the control yeast membrane fraction, were able to activate both CD4 ⁺ and CD8 ⁺ T cells of HIV-infected individuals. We tested the effect of cross-presentation of VLP by DCs in two subjects recruited into a long-term nonprogressor-slow progressor cohort. When yeast VLP-loaded DCs of these patients were cocultured with peripheral blood mononuclear cells for 7 days, approximately one-third of the Gag-specific CD8 ⁺ T cells were activated and became perforin positive. However, some of the Gag-specific CD8 ⁺ T cells appeared to be lost during in vitro culture, especially in a patient with a high virus load. Our results suggest that DCs loaded with yeast VLPs can activate Gag-specific memory CD8 ⁺ T cells to become effector cells in chronically HIV-infected individuals, but there still remain unresponsive Gag-specific T-cell populations in these patients.