RMA-S cells do not express functional TAP, yet they express MHC class I molecules at the cell surface, especially at reduced temperatures (26 degrees C). It is generally assumed that such class I molecules are "empty," devoid of any associated peptide. A radiochemical approach was used to label class I-associated peptides and to determine the extent to which Kb molecules in RMA-S cells are associated with peptides. These studies revealed that at 26 degrees C Kb molecules in RMA-S cells are occupied with self-peptides. Such peptides stably associate with Kb at 26 degrees C but easily dissociate from them at 37 degrees C, suggesting low-affinity interactions between Kb and the associated peptides. At 26 degrees C, at least some of these Kb molecules are stably expressed in a peptide-receptive state on the cell surface, whereas at 37 degrees C they are short lived and are only transiently capable of binding and presenting exogenously supplied OVA 257-264 peptide for presentation to CD8+ Kb-restricted T lymphocytes. Thus contrary to current models of class I assembly in TAP-deficient RMA-S cells, the presumably "empty" molecules are in fact associated with peptides at 26 degrees C. Together, our data support the existence of an alternative mechanism of peptide binding and display by MHC class I molecules in TAP-deficient cells that could explain their ability to present Ag.