An extract of chicken intestine contained neuromedin U-like immunoreactivity (36 pmol/g wet tissue weight). The primary structure of the predominant molecular form (NMU-9), comprising 94% of the total immunoreactivity, was established as: Gly-Tyr-Phe-Phe-Phe-Arg-Pro-Arg- Asn-NH2. This sequence differs from that of pig neuromedin U-8 (NMU-8) by the substitution of Leu3 by Phe and, like the corresponding peptide from the guinea pig, is extended from the NH2-terminus by a Gly residue. A minor component of neuromedin U comprised 25 amino acid residues. An extract of chicken whole brain contained much less NMU-like immunoreactivity (1.5 pmol/g) and the nonpeptide was the only molecular form detected. Synthetic chicken NMU-9 produced a concentration-dependent contraction of smooth muscle from the rat uterus and its effect was unchanged in the presence of tetrodotoxin, atropine and indomethacin. The potency of chicken NMU-9 (EC50 360 +/- 60 nM; mean +/- S.E., n = 6) was approximately 8-fold less than that of pig NMU-8 (EC50 46 +/- 8 nM) but the maximum contraction produced by both agonists was not significantly different.