The Sephadex G-50 gel filtration profile of immunoreactive gastric inhibitory polypeptide (GIP) in porcine and human gastrointestinal mucosa was determined in assays with antisera obtained from five different groups working with GIP. Tissue was extracted in acid ethanol or using a boiling method. Three well-defined components were detected with three of the antisera: one component corresponding to natural porcine GIP (5 kDa GIP), one component corresponding to what has been called 8 kDa GIP, and one component somewhat larger than the latter. One antiserum did not measure 8 kDa GIP at all, while the fifth antiserum measured small amounts of 8 kDa GIP in porcine but not in human extracts. While the antisera measured the same amounts of GIP in porcine extracts, two of the antisera measured significantly more GIP than the remaining three in extracts of human mucosa. The elution position of human 5 kDa GIP differed significantly from that of porcine 5 kDa GIP. In addition to the identification of a new molecular form of GIP, and the demonstration of important specificity differences among GIP antisera currently in use, the present results indicate that human and porcine 5 kDa GIP differ in chemical composition.