Eight antisera were obtained from three rabbits and two guinea pigs immunized with synthetic porcine motilin and from three guinea pigs immunized with [15-Glutamine]-rnotilin. Specificities of the antisera were characterized and three of them having different specificities were used in radioimrnunoassay to examine motilin immunoreactivity in extracts of canine, porcine, human and rat gastrointestinal tissues. Immunoreactive motilin in rat tissue extract showed a very low cross-reaction in all of the assay systems used, suggesting a relatively high degree of variation in the amino acid sequence of rat motilin as compared with that of porcine motilin. In the assay system using the carboxyl-terminal specific antiserum R-1105, the inhibition curves of canine, porcine and human tissue extracts were superimposable on that of synthetic porcine motilin. However, in the assay system using antiserum R-1106 or GP-1103, the inhibition curve of canine tissue extract was not parallel to that of synthetic motilin. Human intestinal tissue extract also showed slightly weak inhibition in the assay system with antiserum R-1106. The motilin concentrations in duodenum extracts of the dog, pig and man measured by four different immunoassay systems were variable depending on the species of the tissue and antiserum used. Gel filtration of human duodenum and jejunum mucosa showed that the tissues contain three major components with motilin-like immunoreactivity. The results suggest either the presence of degradation products of motilin precursor or the existence of a new family of related peptides.KEY WORDS motilin-like immunoreactivity / radioimmunoassay / tissue extracts Bollman fistula, duodenal alkalinization resulted in an increase in serum motilin with a corPorcine motilin is a docosapeptide (1, 15): H-Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-LeuGln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Asn-LysGly-Gln-OH, which has been isolated from the duodenal mucosa (2), In dogs with a denervated pouch of the fundus of the stomach and Mann-