Human maternal tolerance to a semiallogenic fetus may be maintained, in part, by the unusual expression pattern of antigen-presenting molecules in placental trophoblast cells. Extravillous cytotrophoblast (EVC) cells, which invade the maternal decidua, express high levels of human leukocyte antigen G (HLA-G), a nonclassical, major histocompatibility complex (MHC) class I molecule. HLA-G transcripts have been detected in tumors and other tissues, yet protein accumulation is rare. We show that, within EVC cells themselves, the mRNA is more broadly expressed than the protein. Specifically, accumulation of HLA-G protein was markedly delayed during EVC cell differentiation. To elucidate this mechanism, we performed a comprehensive analysis comparing the expression of HLA-G and proteins essential for MHC class I expression at the cell surface. The transporter for antigen processing proteins TAP1 and TAP2, as well as tapasin and beta(2)-microglobulin, appeared to be coordinately expressed throughout EVC cell columns. Strikingly, they all accumulated well in advance of the HLA-G protein but concurrently with its mRNA. A similar delay in the accumulation of the HLA-G protein was observed in vitro, using cultures of chorionic villi. We conclude that posttranscriptional regulation of HLA-G is fundamental to EVC cell development and is achieved independently of the peptide loading system. This represents a novel mechanism of MHC class I regulation.