Dog neuromedin U-8 (d-NMU-8; pGlu-X2-Leu-Y4-Arg-Pro-Arg-Asn-NH2, X = Y = Phe) has potent biological activity to stimulate an isolated chicken crop smooth muscle preparation with the relative activity (RA value) of 5.78 to porcine neuromedin U-8 (p-NMU-8). To elucidate the contribution of the two phenylalanine residues of NMU-8 to the biological activity, fourteen d-NMU-8 analogs modified either at position 2 or 4, [X2]- or [Y4]-d-NMU-8, were synthesized, where X and Y were Ala, Tyr, Trp, Thr, Glu, His or cyclohexylalanine (Cha). Most of the analogs retained very low contractile activity, suggesting the importance of both Phe residues in d-NMU-8 for the biological activity. [X2]-d-NMU-8 analogs had lower biological activity in terms of the RA value than the corresponding [Y4]-d-NMU-8, when X and Y are the same amino acid. Loss of aromaticity of Phe2 ([Cha2]-d-NMU-8) resulted in a marked decrease of the contractile activity, while that of Phe4 ([Cha4]-d-NMU-8) resulted in retention of considerable activity, with the RA value of 2.68. [Tyr4]-d-NMU-8 was an exceptional analog with higher contractile activity (p < 0.01) than the parent compound d-NMU-8, having the RA value of 12.6. The results indicated that the aromatic side chain of the Phe residue at position 2 contributes more than that at position 4 to the biological activity.