The impairment of intestinal perfusion following induction of a pneumoperitoneum may lead to a reduction of peritoneal tissue plasminogen activator (tPA) activity and a concomitant increased risk of adhesion formation. Pigs were laparotomized to take peritoneal biopsy specimens from the cecum, the ileum, and the abdominal wall. A 15 mmHg pneumoperitoneum was established for 3 h by the insufflation of carbon dioxide (group 2, n = 6) or helium (group 3, n = 6). Group 1 (n = 7) received no gas insufflation. After a 2-h recovery period, additional tissue samples were harvested. Specific tPA activity was then determined in the tissue extracts. During surgery, specific tPA activity decreased in all the samples. As compared with the control group (100%), this reduction was strongly aggravated in the cecum (-67.6%, p < 0.05) and the ileum (-70.8%) of the CO2 group but only slightly aggravated in the helium group. The parietal peritoneum was not specifically affected by gas insufflation. The use of a pneumoperitoneum with carbon dioxide significantly affects peritoneal tPA activity and thus may represent a stimulus for adhesion formation.