Viable Escherichia coli and Staphylococcus aureus bacteria elicited markedly different in vitro tumor necrosis factor alpha (TNF-α) responses when placed in coculture with peritoneal murine macrophages. These include quantitative differences in TNF-α mRNA expression and corresponding protein product secretion as well as kinetic differences in the profiles of the TNF-α responses. Further, lipopolysaccharide (from E. coli) is a major contributing factor to these differences, as revealed by comparative experiments with endotoxin-responsive (C3Heb/FeJ) and endotoxin-hyporesponsive (C3H/HeJ) macrophages. Nevertheless, the eventual overall magnitude of the TNF-α secretion of macrophages in response to S. aureus was at least equivalent to that observed with E. coli, while appearing at time periods hours later than the E. coli-elicited TNF-α response. Both the magnitude and kinetic profile of the TNF-α responses were found to be relatively independent of the rate of bacterial proliferation, at least to the extent that similar results were observed with both viable and paraformaldehyde-killed microbes. Nevertheless, S. aureus treated in culture with the carbapenem antibiotic imipenem manifests markedly altered profiles of TNF-α response, with the appearance of an early TNF-α peak not seen with viable organisms, a finding strikingly similar to that recently reported by our laboratory from in vivo studies (R. Silverstein, J. G. Wood, Q. Xue, M. Norimatsu, D. L. Horn, and D. C. Morrison, Infect. Immun. 68:2301–2308, 2000). In contrast, imipenem treatment of E. coli-cocultured macrophages does not significantly alter the observed TNF-α response either in vitro or in vivo. In conclusion, our data support the concept that the host inflammatory response of cultured mouse macrophages in response to viable gram-positive versus gram-negative microbes exhibits distinctive characteristics and that these distinctions are, under some conditions, altered on subsequent bacterial killing, depending on the mode of killing. Of potential importance, these distinctive in vitro TNF-α profiles faithfully reflect circulating levels of TNF-α in infected mice. These results suggest that coculture of peritoneal macrophages with viable versus antibiotic-killed bacteria and subsequent assessment of cytokine response (TNF-α) may be of value in clarifying, and ultimately controlling, related host inflammatory responses in septic patients.