Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers that present peptides on the surface of antigen presenting cells to T cells. Soluble HLA-DR2 molecules were expressed for structural and functional characterization of the MHC/ peptide/T cell receptor recognition unit. The ␣ and ␤ chains of DR2 (encoded by the DRA, DRB1‫1051ء‬ genes) did not assemble in mammalian or insect cell lines when the transmembrane regions of one or both chains were truncated. The hydrophobic transmembrane regions of DR␣ and DR␤ facilitate assembly of the heterodimer and were therefore replaced by the leucine zipper dimerization motifs from the transcription factors Fos and Jun, which assemble as a soluble, tightly packed coiled coil structure. The DR␣-Fos and DR␤-Jun constructs were expressed in a methyltrophic yeast, Pichia pastoris, using the ␣-mating factor secretion signal to direct expression to the secretory pathway. DR ␣␤ heterodimers were purified from supernatants using an antibody specific for the DR ␣␤ heterodimer. Kinetic and quantitative peptide binding experiments demonstrated that recombinant DR2 molecules were efficiently loaded with an antigenic peptide. Soluble DR2 molecules can be used to define structural aspects of the MHC/peptide/T cell receptor interaction and to study the signals induced by T cell receptor recognition of soluble DR2⅐peptide complexes.