Background The group A streptococcus (GAS), Streptococcus pyogenes, is an important cause of both focal and invasive infections. Infections caused by GAS are often associated with severe inflammation. Exposure of macrophages to GAS stimulates the production of inflammatory mediators, including tumor necrosis factor (TNF), and via the inducible nitric oxide synthase (iNOS) enzyme nitric oxide (NO). Several GAS components have been reported to trigger the host inflammatory response, but the role of GAS DNA has not been examined. Purpose We sought to define the role of bacterial DNA in stimulating the macrophage inflammatory response to antibiotic-treated live GAS. Methods RAW 264.7 murine macrophages were stimulated for 16 to 18 hours with 106 or 107 cfu/mL of a well-characterized M1T1 GAS isolate in the presence of penicillin G (5 mg/L). Recombinant interferon-γ 10 U/mL was added for the iNOS experiments. In a series of parallel experiments, cells were preincubated for 1 hour prior to bacterial challenge with one of two selective inhibitors of the bacterial DNA/Toll-like receptor 9 (TLR9) pathway, either chloroquine (2.5 mg/L) or iCpG DNA, an inhibitory oligonucleotide (3 mg/L). TNF concentrations in cell supernatants were determined by ELISA, and accumulation of iNOS protein in cell lysates was quantitated by immunoblotting. Results We found that penicillin-treated GAS stimulated the secretion of large amounts of TNF and the accumulation of substantial amounts of iNOS protein by RAW 264.7 cells. Preincubation of these cells with chloroquine led to significantly reduced secretion of TNF (24-38% less) in response to antibiotic-treated GAS but did not affect iNOS protein accumulation under these conditions. Preincubation of RAW cells with iCpG DNA resulted in a 17% decline in GAS-stimulated TNF secretion (although this difference was not statistically significant) and had no effect on iNOS production. Conclusions Our data suggest that bacterial DNA contributes to the magnitude of the TNF but not iNOS response by macrophages exposed to whole, antibiotic-treated GAS. The role of bacterial DNA in triggering the host response to GAS deserves additional study.