Lipoteichoic acids (LTA), cell wall components of gram-positive bacteria, have been reported to induce various inflammatory mediators and to play a key role in gram-positive-microbe-mediated septic shock. In a large number of these studies, investigators used commercially available LTA purified from a variety of gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, and Streptococcus sanguis. We report here that, although these commercially available LTA could be readily shown to stimulate production of nitric oxide (NO) in RAW 264.7 mouse macrophages, the activity was dramatically inhibited by polymyxin B, a relatively specific inhibitor of endotoxin biological activity. One-step purification of the commercially available S. aureus LTA using hydrophobic interaction chromatography resulted in two well-separated peak fractions, one highly enriched for LTA and a second highly enriched for endotoxin. The LTA-enriched fractions did not induce production of NO in RAW 264.7 macrophages, although they caused a dose-dependent induction of NO in the presence of low concentrations of gamma interferon (IFN-gamma) (which by itself induced little NO), regardless of the presence of polymyxin B. In contrast, the endotoxin-enriched fractions by themselves inhibited in high levels of NO in RAW 264.7 macrophages but activity was almost completely inhibited in the presence of polymyxin B. Consistent with these findings, our data also indicate that commercial LTA preparations from S. aureus, B. subtilis, and S. sanguis were not able to induce NO from lipopolysaccharide-hyporesponsive C3H/HeJ mouse peritoneal macrophages, but in the presence of IFN-gamma, these LTA preparations were able to induce relatively high levels of NO from C3H/HeJ macrophages. These results indicate that commercially available LTA can contain contaminating and potentially significant levels of endotoxin that can be expected to contribute to the putative macrophage-stimulating effects of LTA as assessed by NO production. The fact that the purified LTA, by itself, was not able to induce significant levels of NO secretion in RAW 264.7 macrophages supports the conclusion that caution in attributing high-level biological activity to this microbial cell wall constituent should be exercised.