To persist in the presence of an active immune system, viruses encode proteins that decrease expression of major histocompatibility complex class I molecules by using a variety of mechanisms. For example, murine gamma-2 herpesvirus 68 expresses the K3 protein, which causes the rapid turnover of nascent class I molecules. In this report we show that certain mouse class I alleles are more susceptible than others to K3-mediated down regulation. Prior to their rapid degradation, class I molecules in K3-expressing cells exhibit impaired assembly with beta(2)-microglobulin. Furthermore, K3 is detected predominantly in association with class I molecules lacking assembly with high-affinity peptides, including class I molecules associated with the peptide loading complex TAP/tapasin/calreticulin. The detection of K3 with class I assembly intermediates raises the possibility that molecular chaperones involved in class I assembly are involved in K3-mediated class I regulation.