We have shown that the inflammation-attenuating effects of CO(2) pneumoperitoneum during laparoscopy are not due to changes in systemic pH. However, acidification of peritoneal macrophages in an in vitro CO(2) environment has been shown to reduce LPS-mediated cytokine release. We tested the hypothesis that the peritoneum is locally acidotic during abdominal insufflation with CO(2)--even when systemic pH is corrected. Rats (n = 20) were anesthetized and randomized into two groups: continued spontaneous ventilation (SV) or intubation and mechanical ventilation (MV). All animals were then subjected to abdominal insufflation with CO(2). Mean arterial pH among SV rats decreased significantly from baseline after 15 and 30 minutes of CO(2) pneumoperitoneum (7.329 --> 7.210 --> 7.191, P < 0.05), while arterial pH among MV rats remained relatively constant (7.388 --> 7.245 --> 7.316, P = NS). In contrast, peritoneal pH dropped significantly from baseline and remained low for both groups during CO(2) abdominal insufflation (SV 6.74 --> 6.41 --> 6.40, P < 0.05; MV 6.94 --> 6.45 --> 6.45, P < 0.05). In a second experiment, rats (n = 10) were randomized to receive abdominal insufflation with either CO(2) or helium. Abdominal insufflation with helium did not significantly affect peritoneal pH (7.10 --> 7.02 --> 6.95, P = NS), and the decrease in pH among CO(2)-insufflated animals was significant compared with helium-insufflated animals (P < 0.05). Peritoneal pH returned to baseline levels in all groups within 15 minutes of desufflation in both experiments. A significant local peritoneal acidosis occurs during laparoscopy which is specifically attributable to the use of CO(2) and which is independent of systemic pH. These data provide additional evidence that localized peritoneal acidosis is central to the mechanism of CO(2)-mediated attenuation of the inflammatory response following laparoscopic surgery.