Proneurotensin/neuromedin N (pro NT/NMN) is the common precursor of two biologically active peptides, neurotensin (NT) and neuromedin N (NMN). We have established antibodies against peptide sequences of the NT/NMN precursor and developed a sandwich immunoassay for the detection of pro NT/NMN immunoreactivity in human circulation. Endogenous pro NT/NMN immunoreactivity was enriched by affinity chromatography using antibodies against two different pro NT/NMN epitopes, and further purified by reversed phase HPLC. Mass spectrometry analysis revealed pro NT/NMN 1-117 as major pro NT/NMN immunoreactivity in human circulation. Pro NT/NMN 1-117 is detectable in serum from healthy individuals (n = 124; median 338.9 pmol/L). As known for NT, the release of pro NT/NMN 1-117 from the intestine into the circulation is stimulated by ingestion of an ordinary meal. Investigation of the pro NT/NMN 1-117 in vitro stability in human serum and plasma revealed that this molecule is stable for at least 48 h at room temperature. Since pro NT/NMN 1-117 is theoretically produced during precursor processing in stoichiometric amounts relative to NT and NMN, it could be a surrogate marker for the release of these bioactive peptides.