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The aim of the present study was to examine the presence of motilin in the duodenal epithelial cells of premature infants of <32 weeks gestation.Specimens from 10 deceased infants (gestational age: 26.4 ± 2.7 weeks and birthweight: 808 ± 303 g) were examined as subjects. All infants died of severe cardiopulmonary disorder or intraventricular hemorrhage (grade IV). The average survival period was 3.1 ± 1.9 days. Autopsies were performed and formalin-fixed duodenums were immunostained with rabbit antiserum to motilin by the labeled streptavidin–biotin (LSAB) method. An adult duodenum obtained by pancreatoduodenectomy was also examined for the presence of motilin as a positive control specimen. An absorption test using motilin peptide was performed to prove the specificity of the binding with rabbit antiserum to motilin.Motilin-containing cells were detected in the adult specimen, and the binding by rabbit antiserum to motilin was completely inhibited by excess amounts of motilin peptide, indicating that this binding was specific to motilin. All 10 infants had presence of motilin antigen in the epithelial cells of their duodenums.This preliminary study indicates that the immunohistological analysis is specific to detect motilin-containing cells, and certifies the presence of motilin in duodenal epithelial cells of premature infants of <32 weeks gestation, including one at only 22 weeks gestation.