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-2Kb heavy chain, mouse beta 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 Kb-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 Kb molecules, but generated cell surface Kb-OVA(257-264) complexes at least 20-fold less efficiently from ER-targeted peptides. The inefficient assembly of Kb-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 Kb-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 Kb-OVA(257-264) complexes. The assembly of Kb-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.