Abstract: The aim this study focus on understanding the characteristics of specific amino acid residues located in chicken invariant chain (Ii) transmembrane domain in the formation of MHC ⅡIi complex. Two amino acids, Gln47 and Thr50, in transmembrane region of chicken Ii were substituted by Ala via site mutation by the PCR megaprimer method, then the mutated fragment was inserted into pEGFPC1 vector and the eukaryotic expression vector containing IiGFP fusion gene was constructed. At the same time the other eukaryotic expression plasmids, pDsRed2N1MHC Ⅱα, pDsRed2N1MHCⅡβ, pEGFPN1MHCⅡα and pEGFP N1MHCⅡβ were constructed respectively. These recombinant plasmids were solely or cotransfected into COS7 cells with LipofectamineTM 2000. After culture of the cells for 48 h, the expression and intracellular localization of wild type and mutated Ii and MHC Ⅱsubunits were observed with a fluorescent microscope, and their association was analyzed by an immunoprecipitation test. The results showed that the wild type Ii with MHCⅡα or MHCⅡβ polypeptides could colocalized in cells, while the mutated Ii could not colocalized with MHC Ⅱα or MHC Ⅱβ in the endocytic compartments. The results of immunoprecipitation showed that the wild type Ii could bind MHC Ⅱα or (and) MHC Ⅱβ subunits when pEGFPC1Ii, pEGFPN1MHC Ⅱα or (and) pEGFPN1MHC Ⅱβ were cotransfected in COS7 cells, while the mutated Ii could not colocalized with MHC Ⅱα or (and) MHC Ⅱβ subunits when pEGFPC1IiQ47A (or pEGFPC1IiT50A), pEGFPN1MHC Ⅱα or (and) pEGFPN1MHC Ⅱβ were cotransfected in COS7 cells. Therefore all these results suggest that Gln47 and Thr50 in transmembrane region of chicken Ii play a key role in the assembly of Ii and MHCⅡsubunits.