Loading of peptides onto major histocompatibility complex class I molecules involves a multifactorial complex that includes tapasin (TPN), a membrane protein that tethers empty class I glycoproteins to the transporter associated with antigen processing. To evaluate the in vivo role of TPN, we have generated Tpn mutant mice. In these animals, most class I molecules exit the endoplasmic reticulum (ER) in the absence of stably bound peptides. Consequently, mutant animals have defects in class I cell surface expression, antigen presentation, CD8+ T cell development, and immune responses. These findings reveal a critical role of TPN for ER retention of empty class I molecules. Tpn mutant animals should prove useful for studies on alternative antigen-processing pathways that involve post-ER peptide loading.