The p35 isoform of the human invariant chain (Iip35) contains an N-terminal RXR endoplasmic-reticulum (ER) retention signal that becomes nonfunctional only after assembly with MHC-class-II molecules. We have previously shown that the MHC-class-II beta-chain cytoplasmic tail is crucial for the maturation of class-II/Iip35 complexes. In order to shed some light on the molecular determinants involved in shielding the RXR motif, we performed site-directed mutagenesis of the DRbeta chain and Ii cytoplasmic domains. Chimeric beta chains with irrelevant cytoplasmic tails allowed the efficient transport of Iip35 out of the ER in transiently transfected HEK 293T cells. An alanine scan of the cytoplasmic tail of HLA-DRbeta confirmed that no specific motif is required to overcome ER retention. Surprisingly, a beta chain with a three-amino-acid-long cytoplasmic tail (Tyr-Phe-Arg) was sufficient to overcome the Iip35 RXR motif. Moreover, replacement of residues F231 and R232 with alanines created a cytoplasmic tail (Tyr-Ala-Ala) that allowed ER egress. Given the limited length of this tail, steric hindrance would only be possible if the Ii ER retention motif was close to the membrane in the first place. However, this is not likely because an Ii molecule with an internal cytoplasmic deletion bringing the RXR motif closer to the membrane is not retained in the ER, even in the absence of class-II molecules. These results suggest that MHC-class-II molecules overcome ER retention and prevent COPI binding to the Iip35 RXR motif through a mechanism distinct from steric hindrance by its beta chain.