Generation of CD8 T-cell responses to pathogens and tumors requires optimal expression of class I major histocompatibility complex/peptide complexes, which, in turn, is dependent on host cellular processing events and subject to interference by pathogens. To create a stable structure that is more immunogenic and resistant to immune evasion pathways, we have engineered class I molecules as single-chain trimers (SCTs), with flexible linkers connecting peptide, beta2m, and heavy chain. Herein we extend our earlier studies with SCTs to the K(b) ligand derived from vesicular stomatitis virus (VSV) to characterize further SCTs as probes of immune function as well as their potential in immunotherapy. The VSVp-beta2m-K(b) SCTs were remarkably stable at the cell surface, and immunization with DNA encoding SCTs elicited complex-specific antibody. In addition, SCTs were detected by cytotoxic T-lymphocytes specific for the native molecule, and the covalently bound peptide was highly resistant to displacement by exogenous peptide. SCTs can also prime CD8 T-cells in vivo that recognize the native molecule. Furthermore, SCTs were resistant to downregulation by the immune evasion protein mK3 of gamma herpesvirus 68. Moreover, owing to their preassembled nature, SCTs should be resistant to other immune evasion proteins that restrict peptide supply. Thus, SCTs possess therapeutic potential both for prophylactic treatment and for the treatment of ongoing infection.