BACKGROUND Human Cytomegalovirus (HCMV) has acquired through evolution a number of genes to try to evade immune recognition of the virus-infected cell. Many of these mechanisms act to inhibit the MHC class I antigen presentation pathway, but any virus-infected cell which has down-regulated cell surface expression of MHC class I proteins, to avoid CTL attack, would be expected to become susceptible to lysis by Natural Killer cells. Surprisingly, however, HCMV infected fibroblasts were found to be resistant to NK cell mediated cytotoxicity. Expression of the UL16 glycoprotein could represent one mechanism to help the virus to escape from NK cell attack, as it has been shown to bind, in vitro, some of the ligands for NKG2D, the NK cell activating receptor. Here, we explored the role of UL16, in the context of a viral infection, by comparing the susceptibility to NK lysis of cells infected with HCMV and cells infected with a UL16 deletion mutant of this virus. RESULTS Cells infected with the UL16 knockout virus were killed at substantially higher levels than cells infected with the wild-type virus. This increased killing could be correlated with a UL16-dependent reduction in surface expression of ligands for the NK cell activating receptor NKG2D. CONCLUSIONS Expression of the UL16 glycoprotein was associated with protection of HCMV-infected cells from NK cell attack. This observation could be correlated with the downregulation of cell surface expression of NKG2D ligands. These data represent a first step towards understanding the mechanism(s) of action of the UL16 protein.