Major histocompatibility complex (MHC) class I molecules act as peptide receptors to direct the recognition of foreign antigens by cytolytic T cells. The cell surface expression and trafficking of these peptide receptors is thought to be controlled by the conformation of the MHC molecule and possibly by the phosphorylation of the cytoplasmic portion of the heavy chain protein. It is of some interest that adenoviruses (Ads) have evolved proteins that interfere with the expression of MHC molecules. One of these proteins, called E3/19k, binds to newly synthesized MHC molecules in the rough endoplasmic reticulum (RER) and inhibits their trafficking to the cell surface. Here we show that during the infection of a human cell line with Ad2, the phosphorylation of the endogenous MHC molecules is inhibited. We also observe that the phosphorylation of the endogenous HLA molecules is grossly impaired in a human cell line transfected with the Ad2 EcoRI D fragment containing the E3/19k gene. We conclude that the E3/19k protein inhibits the phosphorylation of the MHC heavy chains and that this may be one of the important functions of this protein in infected cells. In addition, we show that a mutant of the E3/19k protein, which lacks an RER retention signal but which retains its ability to bind to HLA molecules, does not inhibit the phosphorylation of HLA molecules and that phosphorylated molecules are not Endo H sensitive. This suggests that HLA molecules are phosphorylated after leaving the medial-Golgi compartment, thus providing the most compelling evidence yet that HLA molecules are phosphorylated at or near the cell surface. Finally, to our knowledge, this is the first study under which the phosphorylation of MHC molecules is shown to be altered and may have some relevance for other pathogenic conditions.