Leu-enkephalin analogues containing a ureylene bond instead of an amide bond were synthesized. The ureylene bond was formed by a coupling reaction of the isocyanate derived from the corresponding azide by Curtius rearrangement reaction. Three analogues, each of which has a ureylene bond at the Tyr-Gly or Gly-Gly or Phe-Leu amide bond, were prepared. The ureylene bond resisted enzymatic hydrolysis, but the biological activities of the synthetic peptides on guinea pig ileum and mouse was deferens were low compared with those of Leu-enkephalin.