The alpha beta T-cell receptors (TCRs) react with complex ligands composed of peptides bound to major histocompatibility complex (MHC) proteins. In the absence of foreign antigens the peptides bound to MHC molecules come from the proteins of the host itself. Interactions between TCRs and these self-peptide-MHC ligands work positively to drive T-cell development in the thymus and negatively to delete or inactivate T cells with potential self-reactivity. On the cell surface, MHC proteins are associated with many different self peptides, making it impossible to know which self peptide was involved in positive or negative interactions with a particular T cell. These studies as well as in vitro studies on TCR-peptide-MHC interactions would be aided by a means of producing MHC molecules containing a single peptide. We have tackled this problem for MHC class II proteins by genetically attaching the peptide by a flexible peptide linker to the amino terminus of the class II beta-chain. Here we report that a secreted, soluble form of this covalent peptide-MHC complex can be expressed in insect cells. The peptide is engaged by the peptide-binding groove of the secreted MHC molecule and this complex is recognized by T cells bearing receptors specific for that combination.