Abstract The biosynthesis of substance P has been examined by incubation of dorsal root ganglia with [ 35S]methionine and [ 3H]proline, immunoprecipitation with a variety of antisera and analysis of immunoprecipitates by high performance liquid chromatography. Both amino acids were incorporated into a major peak which co-eluted with substance P and, like the synthetic peptide, could be oxidized to a sulphoxide derivative with a shorter retention time. The peptide was immunoprecipitated by both Nand C-terminally directed substance P antisera, including a monoclonal antibody. Substance P biosynthesis was inhibited by cycloheximide and there was a delay of 1–2 h between addition of radiolabelled amino acids and their appearance in substance P, suggesting the existence of a non-immunoreactive polypeptide precursor which is only slowly processed into substance P. Substance P biosynthesis was reduced to <20% of normal levels in ganglia from animals treated neonatally with capsaicin. [ 35S]methionine was incorporated into a second peptide which was immunoprecipitated only by C-terminally directed substance P antisera and was not labelled by [ 3H]proline. It did not correspond with any of the C-terminal fragments of substance P, and may represent a novel peptide similar to substance P in the C-terminal region but with a different amino acid sequence at the N-terminus.