tStaphylococcus aureus is a Gram-positive bacterium that causes inflammation at infection sites by induc-ing various inflammatory mediators such as nitric oxide (NO). To identify the staphylococcal virulencefactors contributing to NO production, we compared the ability of ethanol-killed wild-type S. aureus andmutant strains lacking lipoteichoic acid (ltaS), lipoproteins (lgt), or d-alanine (dltA) to stimulate NOproduction in a murine macrophage cell line, RAW 264.7, and the primary macrophages derived fromC57BL/6 mice. Wild-type, ltaS, and dltA strains induced NO production in a dose-dependent mannerbut this response was not observed when the cells were stimulated with the lgt strain. Moreover, puri-fied lipoproteins triggered NO production in macrophages. Coincident with NO induction, the wild-type,ltaS, and dltA strains induced expression of inducible NO synthase (iNOS) at both mRNA and proteinlevels whereas lgt failed to induce iNOS protein or mRNA. Transient transfection followed by a reportergene assay and Western blotting experiments demonstrated that wild-type, ltaS, and dltA strains, butnot the lgt strain, induced substantial activation of NF-B and STAT1 phosphorylation, both of whichare known to be crucial for iNOS expression. Moreover, wild-type, ltaS, and dltA strains increasedToll-like receptor 2 (TLR2) activation, which is known to mediate S. aureus-induced innate immunity,whereas the lgt strain did not. Collectively, these results suggest that lipoproteins in the cell wall of S.aureus play a major role in the induction of NO production in murine macrophages through activation ofthe TLR2 receptor.