Introduction: The key to the accelerated recovery of laparoscopic versus open surgery has remained unidentified until now. The anti-inflammatory effects of CO2 have been touted by many including ourselves without clarification of the cellular or molecular mechanisms. Using a model of macrophage depletion we have exposed the cellular target of CO2. Methods: Male Sprague-Dawley rats were depleted of their peritoneal macrophages using clodronate liposomes. Control rats received empty liposomes leaving their peritoneal macrophages intact and functional. The macrophage deplete rats and control rats were subdivided into 4 experimental groups: LPS; anesthesia; CO2 or Helium pneumoperitoneum. All experimental groups received bacterial endotoxin immediately following their 30 min experimental procedure. Blood was harvested for determination of cytokine levels of IL-10 and TNF-alpha. Results: The CO2-pneumoperitoneum significantly increased plasma levels of IL-10 in control rats compared to LPS controls (1993 vs 723 pg/ml; p<0.05 n=6). In rats deplete of their peritoneal macrophages the CO2-mediated increase in IL-10 was abrogated to control levels. This effect was specific to CO2. Alternatively, plasma TNF-alpha was attenuated by all experimental conditions compared to LPS control (p<0.005 n=6). However, the attenuating effect of CO2 was significantly greater than that of anesthesia alone (p<0.05). Furthermore, peritoneal macrophage depletion itself significantly decreased TNF-alpha in LPS controls by more than 60% (18630 vs 7189 pg/ml; p<0.005 n=6). Conclusion: These results strongly suggest that the anti-inflammatory effects conveyed by CO2-pneumoperitoneum act via modulation of the peritoneal macrophage response to an inflammatory insult. It is likely that elevated levels of IL-10 provoked by CO2-induced modulation of peritoneal macrophage function contribute to the improved clinical outcome of patients benefiting from minimally invasive procedures such as cholecystectomy.