Abstract CD99 and CD99-like-2 protein (CD99L2) are membrane glycoprotein leukocyte antigens that do not belong to any known protein family. They are expressed on leukocytes and at the endothelial cell junction, and participate in leukocyte diapedesis process. Based upon the similarity in expression pattern, we tested whether they would interact each other, and show here that the interaction of the two molecules is mediated via cytoplasmic domain of both molecules. In bimolecular fluorescence complementation (BiFC) assay, co-expression of CD99 and CD99L2 emitted fluorescence suggesting the interaction between the two molecules and heterodimer formation. The interaction was further confirmed by fluorescence resonance energy transfer (FRET) assay. Previously, we have reported that CD99 forms CD99-CD99 homodimer. Unlike CD99, CD99L2 does not form homodimer, rather it binds to CD99 and forms CD99-CD99L2 heterodimer. CD99-CD99 homodimer and CD99-CD99L2 heterodimer exist in a cell without competition. With cytoplasmic domain mutant of CD99, CD99L2 could not form heterodimer, indicating two molecules join through cytoplasmic domain. In reverse case when cytoplasmic domain mutant of CD99L2 was used for BiFC, CD99 could not form heterodimer. The interaction between CD99 and CD99L2 was verified by co-immunoprecipitation using mouse spleen cells. Our results reveal that mouse CD99 interact with CD99L2 and form heterodimer through its cytoplasmic domain.