In human neutrophils, interferon (IFN)-γ enhanced the expression of toll-like receptor 4 (TLR4), a crucial component of the signaling receptor complex for bacterial lipopolysaccharide (LPS). Lipopolysaccharide alone did not affect TLR4 expression, but costimulation with IFN-γ and LPS induced higher levels of TLR4 expression than stimulation with IFN-γ alone. Using the protein synthesis inhibitor cycloheximide and measuring the expression of CD35 in neutrophils stimulated with IFN-γ and LPS alone or in combination, we could demonstrate that IFN-γ enhances TLR4 by de novo protein synthesis, whereas the addition of LPS acts synergistically by enhancing vesicular mobilization to the cell surface. Costimulation with IFN-γ and LPS induced neutrophil activation and enhanced secretion of the cytokines, interleukin (IL)-8, IL-1β, tumor necrosis factor-α, and IL-12 p70, and phagocytosis of latex beads, processes that were blocked by a monoclonal antibody specific for TLR4. These data suggest that IFN-γ primes neutrophils to respond to LPS.