AbstractHLA‐G is a class Ib (non‐classical) major histocompatibility complex (MHC) protein expressed at the maternal‐fetal interface that inhibits natural killer (NK) cell‐mediated lysis in an allotype‐independent manner. Here we report that the spontaneous endocytosis of HLA‐G is severely reduced because of its short cytoplasmic tail. Class I (classical) MHC proteins on the surface of B cell trans‐fectants detected by primary and secondary antibodies underwent endocytosis at a moderate rate, whereas HLA‐G, chimeric proteins consisting of the extracellular domains of HLA‐C with the C‐terminal sequence of HLA‐G, or glycophosphatidylinositol‐tailed HLA‐C proteins, were not efficiently internalized. In addition, a mutant of β2‐microglobulin (Ser88Cys) that could be specifically labeled with Texas red (or other fluorescent probes) and exchanged into class I or class Ib MHC proteins was employed to study spontaneous internalization of MHC proteins by a non‐perturbative method independent of an antibody ligand. These data are discussed in terms of both the role of HLA‐G expressed on the fetal trophoblast and the function of the cytoplasmic tail in class I MHC proteins.