Endoplasmic reticulum (ER)-associated aminopeptidase (ERAP)1 has been implicated in the final proteolytic processing of peptides presented by major histocompatibility complex (MHC) class I molecules. To evaluate the in vivo role of ERAP1, we have generated ERAP1-deficient mice. Cell surface expression of the class Ia molecules H-2K^b and H-2D^b and of the class Ib molecule Qa-2 was significantly reduced in these animals. Although cells from mutant animals exhibited reduced capacity to present several self- and foreign antigens to K^b-, D^b-, or Qa-1^b–restricted CD8⁺ cytotoxic T cells, presentation of some antigens was unaffected or significantly enhanced. Consistent with these findings, mice generated defective CD8⁺ T cell responses against class I–presented antigens. These findings reveal an important in vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules.