Human cytomegalovirus (HCMV) infection causes down-regulation of major histocompatibility complex class I heavy chains. We determined previously that there are two HCMV loci which encode functions responsible for that phenotype and that US11 is one of these loci (T. R. Jones, L. A. Hanson, L. Sun, J. S. Slater, R. M. Stenberg, and A. E. Campbell, J. Virol. 69:4830-4841, 1995). Through the construction and analysis of defined viral mutants and stably transfected cell lines, we identify US2 as the other locus. US2 is expressed from very early through late times postinfection, with its predominant product being a relatively unstable 24-kDa endoglycosidase H-resistant glycoprotein. In cell lines constitutively expressing US2, free class I heavy chains are proximal targets for US2-induced degradation, shortly after their synthesis. Both US2 and US11 can function in concert with US3 to down-regulate class I. Beta-2-microglobulin-associated heavy chains which are retained in the endoplasmic reticulum as a result of binding to the US3 glycoprotein are susceptible to destabilization caused by both US2 and US11 gene products. Thus, three HCMV genes which affect either the stability or the transport of class I heavy chains have been identified. The observation that each of these proteins is most abundant early in the replicative cycle suggests that they may play an important immunomodulatory role in vivo prior to productive infection, either during the latent or persistent phase or during reactivation.