The carboxy-terminal region of major histocompatibility complex class I (MHC I) molecules is required for the rapid internalization mediated by Kaposi's sarcoma-associated herpesvirus (KSHV) proteins K3 and K5. The cytoplasmic tail of MHC I contains highly conserved serine phosphorylation sites that have been implicated in intracellular trafficking. Indeed, in vivo labeling experiments reveal a lack of MHC I phosphorylation in K5-transfected HeLa cells. Phosphorylation of the MHC I tail was restored upon mutation of the PHD/LAP domain of K5. However, deletion and mutation studies of the MHC I tail show that both K3 and K5 are able to downregulate MHC I lacking the conserved phosphorylation site. This result suggests that inhibition of phosphorylation reflects, but does not cause, MHC I internalization. Interestingly, K3 and K5 differ from each other, as well as from human immunodeficiency virus nef, with respect to the minimal MHC I tail sequences required for MHC downregulation. These data support the notion that K3 and K5 downregulate MHC I molecules by a distinct molecular mechanism that is different from other viral immune evasion molecules.