Microbial stimuli activate cells of the innate immune system by triggering Toll-like receptors (TLR). Activation of macrophages and dendritic cells is further enhanced by secondary signals like IFN-γ. Here we analyzed the interplay of IFN-γ and TLR signaling in cells of the innate immune system. Using a STAT1-dependent reporter construct we show that IFN-γ signaling can be enhanced as well as inhibited by simultaneous stimulation with either defined TLR agonists or whole-bacterial lysates. Short costimulation resulted in the amplification of IFN-γ signaling and was attributable to the p38 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of signal transducer and activator of transcription (STAT)1 on serine 727. In contrast, prolonged co-incubation as well as pre-incubation with TLR agonists led to an inhibition of IFN-γ signaling. TLR triggering induced expression of suppressor of cytokine signaling (SOCS)-1, SOCS-3 and cytokine-inducible SH2 domain-containing protein (CIS). Overexpression of SOCS-1 and, to a lesser extend, of SOCS-3 and CIS inhibited IFN-γ signaling as measured by activation of STAT1. Moreover, pre-incubation with TLR-dependent stimuli impaired IFN-γ-induced MHC class II regulation but enhanced CD40 and CD86 expression. Taken together, the results indicate a tight interplay between TLR and IFN-γ signaling pathways which involve induction of SOCS proteins and serine phosphorylation of STAT1.