Stat1 is phosphorylated on serine 727 within its transactivating domain (TAD) in response to interferons or other immunological signals. We generated gene-targeted mutant mice expressing a serine727-alanine mutant of Stat1. These animals showed increased mortality upon infection with Listeria monocytogenes and impaired clearance of the bacteria from spleen and liver. The Stat1S727A mice were more resistant to the LPS-induced septic shock syndrome, suggesting that Stat1 serine phosphorylation promotes inflammatory responses. Expression of IFN-gamma-induced genes was strongly reduced in macrophages expressing Stat1(S727A). While mutation of Stat1 at S727 did not reduce its binding to chromatin, association with the coactivator CBP and histone acetylation at the interferon-responsive GBP promoter was strongly reduced, suggesting defective recruitment of histone acetylases as the mechanism underlying IFN-gamma hyporesponsiveness. Our data demonstrate that the increase in transcription factor activity caused by Stat serine phosphorylation contributes to macrophage activation and to IFN-gamma-dependent immune responses in vivo.