As a preliminary step towards the use of cell surface single-chain class I major histocompatibility complex (MHC) molecules as T cell immunogens, we have engineered a recombinant gene encoding a full-length cell surface single-chain version of the H-2Dd class I MHC molecule (SC beta Ddm) which has beta 2-microglobulin (beta 2m) covalently linked to the amino terminus of a full-length H-2Dd heavy chain via a peptide spacer. The single-chain protein is correctly folded and stably expressed on the surface of transfected L cells. It can present an antigenic peptide to an H-2Dd-restricted antigen-specific T cell hybridoma. When expressed in peptide-transport-deficient cells, SC beta Ddm can be stabilized and pulsed for antigen presentation by incubation with extracellular peptide at 27 degrees or 37 degrees C, allowing the preparation of cells with single-chain molecules that are loaded with a single chosen antigenic peptide. SC beta Ddm can be stably expressed in beta 2m-negative cells, showing that the single-chain molecule uses its own beta 2m domain to achieve correct folding and surface expression. Furthermore, the beta 2m domain of SC beta Ddm, unlike transfected free beta 2m, does not rescue surface expression of endogenous class I MHC in the beta 2m-negative cells. This strict cis activity of the beta 2m domain of SC beta Ddm makes possible the investigation of class I MHC function in cells, and potentially in animals, that express but a single type of class I MHC molecule.