Developmentally regulated expression of the products of the major histocompatibility complex (MHC) is thought to play a key role in maternal tolerance of the fetal allograft. Here we analyze a cell line (EE2H3), derived from early post‐implantation‐stage mouse embryos, that is defective for MHC class I assembly. To follow expression of a single well‐defined class I product, we introduced the H‐2D^d gene under control of the human β‐actin promoter. We found that the transfected EE2H3 cells expressed abundant levels of H‐2D^d heavy chains and β₂‐microglobulin protein, but only small amounts of H‐2D^d surface protein. Surface expression was rescued by the addition of an appropriate antigenic peptide, or by culturing the cells at low temperature. The phenotype exhibited by EE2H3 is thus remarkably similar to that described for class I‐negative somatic cell variants selected using antibodies and complement. However, a striking difference was that surface expression in H‐2D^d‐transfected EE2H3 cells was markedly enhanced in response to treatment with interferon. Thus, we have identified a novel class I assembly‐defective cell line. Considering that EE2H3 was established from primary cultures of mouse embryo cells without immunoselection, and is therefore likely to represent a cell population normally present in post‐implantation‐stage embryos, these findings raise the possibility that expression of class I surface antigens during early development may in part be controlled post‐translationally at the level of MHC class I assembly.