HLA-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.