HIV-1 is taken up by dendritic cells (DC) and degradation is detectable within a few hours. Little is known about how rapidly HIV-peptide-loaded MHC-II complexes appear on the surface of DC, however. A key impediment to the detailed understanding of MHC-II antigen presentation of HIV-1 has been the lack of good tools to quantitatively measure antigen presentation. We have developed HIV-1-gag p24 and reverse transcriptase (RT)-specific CD4(+) T cell hybridomas that demonstrate high sensitivity and specificity to detect HIV-1 antigens presented by MHC-II on human DC. We demonstrate that ex vivo primary blood myeloid DC (mDC) and monocyte-derived DC (MDDC) presented HIV-1 peptides on MHC-II molecules within 2 h of exposure to virions. HIV-1 was degraded in a compartment requiring acidification for processing and then was loaded onto newly synthesized MHC-II molecules for presentation.