The source of CA125 synthesis is still debated. Endometrial, peritoneal, ovarian and amniotic cells have been demonstrated to produce and secrete CA125. Different studies show that the peritoneum is a source of CA125. The present study aimed at investigating in vivo and in vitro the peritoneal contribution to circulating CA125. Cultures of uterine peritoneum, abdominal peritoneum and myometrium explants were performed and CA125 measured in the culture medium. To modulate the potential production of CA125, the explants were cultured with or without cycloheximide, bacterial lipopolysaccharide (LPS) or ascitic fluid. In a prospective study, we compared a group of patients after abdominal surgery (n = 19; nine men, 10 women) with a group after extra-abdominal surgery (n = 21; 11 men, 10 women), in order to detect a postoperative increase of serum CA125. De-novo synthesis of CA125 could not be demonstrated in the cultures of uterine and abdominal peritoneum and in myometrium, but CA125 concentrations were detectable in the culture medium without being modulated by cycloheximide, LPS or ascitic fluid. After peritoneal surgery, the proportion of patients with increased serum CA125 was significantly higher (P < 0.03) after abdominal surgery as compared with extra-abdominal surgery. This is considered as indirect evidence for in-vivo production of CA125 by the peritoneum.