Presentation of antigenic peptides to T lymphocytes by MHC class I molecules is regulated by events involving multiple endoplasmic reticulum proteins, including tapasin. By studying the effects of substitutions in the tapasin Ig-like domain, we demonstrated that H-2L(d)/tapasin association can be segregated from reconstitution of folded L(d) surface expression. This finding suggests that peptide acquisition by L(d) is influenced by tapasin functions that are independent of L(d) binding. We also found that the presence of a nine-amino acid region in the Ig-like domain of mouse or human tapasin is required for association with L(d), and certain point substitutions in this sequence abrogate human, but not mouse, tapasin association with L(d). These data are consistent with a higher overall affinity between L(d) and mouse tapasin compared with human tapasin. In addition, we found that other point mutations in the same region of the tapasin Ig-like domain affect MHC class I surface expression and Ag presentation. Finally, we showed that the cysteine residues in the Ig-like domain of tapasin influence tapasin's stability, its interaction with the MHC class I H chain, and its stabilization of TAP. Mutagenesis of these cysteines decreases tapasin's electrophoretic mobility, suggesting that these residues form an intramolecular disulfide bond. Taken together, these results reveal a critical role for the tapasin Ig-like domain in tapasin function.