Amino acid sequences of three canine bombesin-like peptides were determined after sequential purification of an extract obtained from 820 g of intestinal muscle. These three peptides contained 27, 23, and 10 amino acid residues. The sequences of the two shorter forms were identical to the corresponding carboxyl-terminal sequence of the heptacosapeptide. The sequence of the largest peptide is H2N-Ala-Pro-Val-Pro-Gly-Gly-Gln-Gly-Thr-Val-Leu-Asp-Lys-Met-Tyr-Pro-Arg-Gly-Asn - His(Trp-Ala-Val-Gly-His-Leu-Met-CONH2). The sequence of the 23-residue peptide is H2N-Gly-Gly-Gln-Gly-Thr-Val-Leu-Asp-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala- Val-Gly(His-Leu-Met-CONH2). The sequence of the decapeptide is: H2N-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-CONH2. Comparison of the 27-residue peptide with the known structure of porcine gastrin-releasing peptide, another bombesin-like heptacosapeptide, reveals four amino acid substitutions: canine bombesin-like peptide had Pro 4, Gly 5, Gln 7, Asp 12, whereas porcine gastrin-releasing peptide had Ser 4, Val 5, Gly 7, Ala 12. Radioimmunoassay of brain extracts after similar purification revealed the presence of similar large and small forms of immunoreactive bombesin peptides, but with apparent tissue concentrations of about 4% of those present in intestinal muscle. Canine bombesin-like peptides represent another example of mammalian neuropeptides existing in more than one biologically active molecular form.