We report a Sendai virus (SeV) vector system for expression of major histocompatibility complex (MHC) class I/peptide complexes. We cloned the extracellular domain of a human MHC class I heavy chain, HLA-A*2402, and human beta-2 microglobulin (beta2m) fused with HLA-A*2402-restricted human immunodeficiency virus type 1 (HIV-1) cytotoxic T-lymphocyte (CTL) epitopes (e-beta2m) in separate SeV vectors. When we coinfected nonhuman mammalian cells with the SeVs, naturally folded human MHC class I/peptide complexes were secreted in the culture supernatants. Biotin binding peptide sequences on the C terminus of the heavy chain were used to tetramerize the complexes. These tetramers made in the SeV system recognized specific CD8-positive T cells in peripheral blood mononuclear cells of HIV-1-positive patients with a specificity and sensitivity similar to those of MHC class I tetramers made in an Escherichia coli system. Solo infection of e-beta2m/SeV produced soluble e-beta2m in the culture supernatant, and cells pulsed with the soluble protein were recognized by specific CTLs. Furthermore, when cells were infected with e-beta2m/SeV, these cells were recognized by the specific CTLs more efficiently than the protein pulse per se. SeV is nonpathogenic for humans, can transduce foreign genes into nondividing cells, and may be useful for immunotherapy to enhance antigen-specific immune responses. Our system can be used not only to detect but also to stimulate antigen-specific cellular immune responses.