In mammalian spinal cord, we have previously discovered "neuromedin B", whose structure is closely related to amphibian bombesin. By utilizing a specific radioimmunoassay for neuromedin B, we have isolated two novel "big" neuromedin B, designated neuromedin B-32 and B-30, from pig brain and spinal cord, both of which were identified as N-terminally extended forms of neuromedin B. The amino acid sequence of neuromedin B-32 was determined to be: Ala-Pro-Leu-Ser-Trp-Asp-Leu-Pro-Glu-Pro- Arg-Ser-Arg-Ala-Gly-Lys-Ile-Arg-Val-His-Pro-Arg-Gly-Asn-Leu-Trp-Ala- Thr-Gly-His-Phe-Met-NH2, while neuromedin B-30 was found to be an N-terminal two amino acids deleted form of neuromedin B-32. Isolation of a family comprising neuromedin B, B-30 and B-32 is indicative of their biosynthetic relationship.