Major histocompatibility complex (MHC) class II molecules are heterodimers of alpha and beta subunits that associate intracellularly with the invariant chain. Human invariant chain exists in four forms, p33, p35, p41, and p43, generated by a combination of alternative initiation of translation and alternative splicing. The biological significance of the existence of the different forms of invariant chain is still unclear and to date no study has compared all four using one system. We have compared them for their transport characteristics and for their ability to transport associated MHC class II heterodimers into the endocytic pathway. Here we report that hetero oligomers containing p33 and p35 or p41 and p43 remain in the endoplasmic reticulum (ER) in the absence of class II alpha and beta chains. This is consistent with earlier reports suggesting that the N-terminal extension shared by p35 and p43 contains an ER retention signal. Homo oligomers containing only the p33 or p41 forms of invariant chain exit the ER and are sorted to endosomes following passage through the Golgi apparatus. Their accumulation leads to enlargement of the endosomes. Quantitation of the turnover rates of the p35/p33 forms with the alternatively spliced p43/p41 forms indicates that the latter are more stable, both in the ER and following transport through the Golgi apparatus. When class II molecules are co-expressed with p33 and p35, or p41 and p43, the assembled complex is efficiently transported to the endocytic pathway.