An in vitro model of human CMV infection of primary retinal pigment epithelial (RPE) cells was used to study the effects of cytokines on CMV replication in these cells, which are targets of CMV infection in vivo. IFN-γ and IFN-β were potent inhibitors of CMV replication in RPE cells, while TNF-α, IL-1β, or TGF-β2 did not affect viral replication. Inhibition by IFN-γ, and to a lesser extent IFN-β, was almost completely reversed by addition of l-tryptophan to the culture medium, strongly implicating the indoleamine 2, 3 dioxygenase (IDO) pathway. Polyadenylated IDO mRNA accumulation was detected as early as 2 h after IFN stimulation. Furthermore, CMV blocked the production of nitric oxide by the inducible form of nitric oxide synthase. This inhibition depended on a functional viral genome. However, exogenous nitric oxide significantly inhibited viral protein expression in RPE cells. Thus, CMV infection blocks the inducible nitric oxide synthase pathway activated by IFN-γ and IL-1β, but cannot counteract the IFN-induced IDO pathway, which ultimately controls its replication in primary human RPE cells.