Virus-specific CD4+ T-cells are essential for maintaining an effective immune response by providing “help” to B-cells and CD8+ T-cells. They may also have a direct cytolytic activity against infected cells. The T cell receptor (TCR) on the surface of human CD4+ T-cells recognises antigenic peptides presented by the class II HLA molecules, HLA-DR, -DQ or -DP. Recognition of these peptide-MHC complexes by CD4+ helper T-cells facilitates B-cell activation, proliferation and the secretion of virus-specific antibody. Most of the HIV epitopes recognised by CD4+ T-cells have been identified by measuring T-cell responses against overlapping peptide libraries. In order to identify the naturally processed and presented forms of HIV envelope derived peptide antigens, HLA class II molecules were immunoaffinity purified from the surface of HIV+ antigen presenting cells and their peptide cargo sequenced by high-resolution mass spectrometry. Using this approach, seventeen epitopes were identified from HIV antigens, of which nine are novel. Four of these novel epitopes span the ectodomain near the N-terminus and are part of the Heptad repeat region 1 which plays a crucial part in viral entry into host cell. A nested set of five epitopes are located on the cytoplasmic tail of envelope and covers a site that is involved in NFKB activation. The epitopes identified will help widen the search for T helper determinants that drive antibody production in vivo. In addition to the HIV peptides, more than 30000 endogenous HLA class II ligands were identified. This represents the largest data set of class II bound peptides generated to date and has provided new insights into the origin and properties of naturally presented HLA class II ligands. This large dataset of HLA-bound peptides will also enable development of more accurate prediction algorithms that will aid in development and identification of CD4+ determinants in HIV and other diseases.