Pseudomyxoma peritonei, a syndrome first described by Rokitansky in 1842, is an enigmatic, often fatal intra-abdominal disease characterized by dissecting gelatinous ascites and multifocal peritoneal epithelial implants secreting copious globules of extracellular mucin. Although much past interest in the syndrome has focused on the question of whether the disease arises from primary appendiceal or ovarian mucinous tumors of varying malignant potential, the accumulation of extracellular mucin with its resulting obstruction of abdominal viscera and adhesion formation is one major cause of this disease's morbidity and mortality irrespective of the origin or transformed status of the epithelium secreting it. Because of this and because of the recent discovery and cloning of a series of specific mucin genes responsible for mucin secretion and extracellular deposition, we decided to analyze cases of pseudomyxoma peritonei with specific mucin cDNAs and corresponding antibodies to identify a characteristic marker for this disease which ultimately might be targeted therapeutically. Our study specifically investigated MUC2 and MUC5AC because these two mucins possessed the physicochemical property of being gel-forming, a property exhibited by pseudomyxoma peritonei grossly. Expression of MUC2 and MUC5AC in pseudomyxoma peritonei and in accompanying and non-accompanying appendiceal and ovarian mucinous neoplasms were analyzed by in situ hybridization, immunocytochemistry and digital image analysis. A striking overexpression of both MUC2 and MUC5AC was observed in nearly all cases of pseudomyxoma peritonei of unknown and appendiceal origin. In these cases, however, MUC2 gene expression was more prominent. The mucin:cell ratio averaged 10 to 1 in these cases. The primary ovarian mucinous tumors, some of which exhibited pseudomyxoma ovarii and/or peritoneal implants but not classic pseudomyxoma peritonei, in contrast, expressed only MUC5AC and gave rise to implants where the mucin:cell ratio averaged only 1 to 1. MUC2 overexpression then supported an intestinal rather than ovarian origin for true pseudomyxoma peritonei, irrespective of whether an appendiceal primary was documented. In all cases studied, the fidelity of MUC2 and MUC5AC expression held irrespective of the degree of malignant transformation which was present. MUC2 is therefore a reliable molecular marker for pseudomyxoma peritonei.