NK cells play a key role in host defense against the beta-herpesvirus CMV through perforin-dependent cytolysis. In this study, we show that human NK cells can also control human CMV (HCMV) infection by a noncytolytic mechanism involving induction of IFN-beta in the virus-infected cell. Both IL-2-activated primary NK cells and an IL-2-dependent NK cell line (NK-92) exhibited potent, noncytolytic anti-HCMV activity at very low E:T cell ratios (<0.1:1). Activated NK cells expressed lymphotoxin (LT)alphabeta on their cell surface, and secreted LTalpha and TNF, all of which contributed to the NF-kappaB-dependent release of IFN-beta from infected fibroblasts. IFN-beta produced by fibroblasts and NK cell-produced IFN-gamma combined to inhibit HCMV replication after immediate early gene expression. These results highlight an efficient mechanism used by NK cells to activate IFN-beta expression in the infected target cell that contributes to the arrest of virion production and virus spread without cellular elimination.