Peptostreptococcus micros is a gram-positive bacterium that has been associated with periodontitis and endodontic infections. In this study, we hypothesized that P. micros binds the immunomodulating component lipopolysaccharide derived from gram-negative bacteria to increase its capacity to stimulate cytokine production by host cells. The ability of P. micros to bind Actinobacillus actinomycetemcomitans lipopolysaccharide was demonstrated by an enzyme-linked immunosorbent assay and by immunoelectron microscopy. Pretreatment of P. micros cells with A. actinomycetemcomitans lipopolysaccharide was associated with a 49-fold increase in tumor necrosis factor alpha production by human monocytic cells U937 differentiated into adherent macrophages, compared to the stimulation with untreated P. micros. This effect was suppressed by incorporating polymyxin B, a lipid A-binding substance, during treatment of macrophage-like cells with lipopolysaccharide-coated P. micros cells. This is the first study reporting a binding interaction between lipopolysaccharide and a gram-positive bacterium. This interaction represents a new mechanism that could promote the inflammatory response during periodontitis.