Systemic administration of adenovirus and adenovirus vectors induces a robust innate and adaptive immune response in a variety of animal models. In tumor necrosis factor (TNF)(-/-) mice, a diminished immune response to adenovirus (Ad) infection has been attributed to compromised dendritic cell (DC) maturation. In this report, we investigated the mechanisms responsible for Ad-mediated activation and maturation of DC. Ad infection induced high levels of TNF-alpha expression by murine bone marrow-derived DC, comparable to levels observed with lipopolysaccharide exposure. Ad-induced TNF-alpha production was necessary for DC maturation and acts in an autocrine manner. Unlike TNF-alpha production associated with exposure to lipopolysaccharide, Ad induction of TNF-alpha was not dependent on the MyD88 signaling pathway. In contrast, Ad-induced TNF-alpha production and DC maturation were dependent on signaling by phosphoinositide-3-OH kinase (PI3K), as determined by wortmannin and LY294002 blocking experiments. The adenovirus capsid protein penton contains a well characterized arginine-glycine-aspartic acid integrin-binding domain that stimulates PI3K in fibroblast cell lines. When this region of the penton was mutated, TNF-alpha expression and bone marrow-derived DC maturation were attenuated. We propose that integrin-mediated PI3K induction of NF-kappaB activates an autocrine TNF-alpha pathway required for DC maturation in response to Ad.