The lumen of the human colon is heavily colonized with microbes, but infections across its epithelial surface are infrequent. To address the hypothesis that antimicrobial polypeptides contribute to the barrier function of colonic epithelial cells, we examined cellular extracts from non-inflamed colonic mucosa using an antimicrobial assay. This approach yielded five polypeptides: three antimicrobials were previously identified as ribosomal polypeptides (L30, S19 and ubiquicidin), and two were members of the histone family (H1.5 and H2B). All exhibited bactericidal activity against Escherichia coli, and with the exception of S19, had been isolated by others based on their potent antimicrobial activity in other cells and tissues. These polypeptides normally reside inside cells and are proposed to contribute to the formation of the functional antimicrobial barrier of the colonic epithelium.