To study the requirements for assembly of MHC class I molecules with antigenic peptides in the endoplasmic reticulum (ER), we studied Ag processing in insect cells. Insects lack a class I recognition system, and their cells therefore provide a “blank slate” for identifying the proteins that have evolved to facilitate assembly of class I molecules in vertebrate cells. H-2K b heavy chain, mouse β 2-microglobulin, and an ER-targeted version of a peptide corresponding to Ova 257–264 were expressed in insect cells using recombinant vaccinia viruses. Cell surface expression of K b-OVA 257–264 complexes was quantitated using a recently described complex-specific mAb (25-D1. 16). Relative to TAP-deficient human cells, insect cells expressed comparable levels of native, peptide-receptive cell surface K b molecules, but generated cell surface K b-OVA 257–264 complexes at least 20-fold less efficiently from ER-targeted peptides. The inefficient assembly of K b-OVA 257–264 complexes in the ER of insect cells cannot be attributed solely to a requirement for human tapasin, since first, human cells lacking tapasin expressed endogenously synthesized K b-OVA 257–264 complexes at levels comparable to tapasin-expressing cells, and second, vaccinia virus-mediated expression of human tapasin in insect cells did not detectably enhance the expression of K b-OVA 257–264 complexes. The assembly of K b-OVA 257–264 complexes could be greatly enhanced in insect but not human cells by a nonproteasomal protease inhibitor. These findings indicate that insect cells lack one or more factors required for the efficient assembly of class I-peptide complexes in vertebrate cells and are consistent with the idea that the missing component acts to protect antigenic peptides or their immediate precursors from degradation.