The invariant chain (Ii) has for many years been known to have important functions in MHC class II antigen presentation. There are four isoforms of Ii in humans: Iip33, Iip35, Iip41 and Iip43. The two shorter isoforms (Iip33 and Iip35) are formed due to two alternative translational start sites, and Iip35 contains an N-terminal extension of 16 aa compared to Iip33. This 16 aa N-terminal extension contains a strong ER retention motif. The Iip41 and Iip43 isoforms have the same N-terminal ends as Iip33 and Iip35 respectively, but in addition they contain an extra exon. Being the predominant isoform Iip33 is studied most extensively, while limited data is available on the human specific isoforms.In this work both imaging and different biochemical techniques were used to study Iip33 and Iip35, both individually and co-expressed. The Ii isoforms associate randomly into heterotrimers in the endoplasmic reticulum (ER). Our results indicate that the two isoforms behave differently when co-expressed, compared to when expressed alone. Iip33 function is highly affected by the presence of Iip35. Iip35 leads to ER retention of Ii heterotrimers. Consequently Iip33 trafficking through the secretory pathway is delayed, and its half-life is significantly increased in the presence of Iip35. The formation of Ii-induced enlarged endosomal vesicles (ILEVs) which is normally observed upon over-expression of Iip33, is not observed upon co-expression with Iip35. Iip35 is also affected by the presence of Iip33 as the half-life of Iip35 is decreased in the presence of Iip33. This suggests a more efficient endosomal sorting of Iip35 in the presence of Iip33.Our results indicate that Iip35 has a large influence on the total pool of Ii even when present in low levels. The properties of Iip35 and the effects of its presence in Ii heterotrimers are thus important to understand MHC class II antigen presentation in humans.