MHC class I molecules assemble within the endoplasmic reticulum (ER) in complexes that include β2-microglobulin (β2m), the transporter associated with antigen processing (TAP) and several additional chaperones. Release of class I complexes from the ER is thought to require the binding of an appropriate endogenous peptide, predominantly delivered from the cytosol to the ER by TAP. It was recently demonstrated that exogenous synthetic peptide could 'directly' enter the ER of intact cells, independently of TAP function, and bind to the class I molecule H-2Kb. In TAP-deficient cells, we show that nascent Kb or Kb-Ld chimeric molecules have a high trafficking background; 50-80% of these class I molecules are released from the ER independently of TAP function or the addition of exogenous peptide. The addition of exogenous Kb cognate peptides enhanced the release of these class I molecules only slightly over the high background. The chimeric class I-b molecule, M3-Ld, differs from Kb-Ld only in its peptide binding domains, and M3-Ld preferentially binds N-formylated peptides, which are rare in eukaryotic cells. Release of M3-Ld from the ER in the absence of exogenous peptide was negligible. Addition of exogenous formylated peptides induced significant trafficking and surface expression of M3-Ld. These observations suggest that peptide binding is necessary for class I release from the ER even in TAP-deficient cells. These results demonstrate that exogenous peptide not only enters the ER of intact cells independently of TAP but also functionally induces class I antigen presentation.