Neurotensin (NT) is coexpressed with catecholamines in sympathetic neurons and adrenal chromaffin cells. A pheochromocytoma PC12 cell line can also be induced to express the NT gene and produce immunoreactive NT. In the present study, NT mRNA was quantified under various hormonal conditions and NT precursor synthesis rates were determined by pulse labeling and immunoprecipitation. In addition, NT precursor and NT-related products were measured using RIA and were characterized using HPLC and Sephadex chromatography. Neurotensin mRNA, NT precursor synthesis, and NT precursor/product levels were correlated. Surprisingly, NT appeared to be a minor product, both in cells and media: NT precursor (approximately 88%), NT(3-13)-like peptide (approximately 10%), and NT (approximately 2%). Neurotensin added to cultures was not converted to NT(3-13). Treatment of cells with 60 mM KCl or various secretagogues induced Ca(2+)-dependent release of NT precursor, NT(3-13), and NT in proportion to their cellular contents. These results suggest a) that NT precursor processing in induced PC12 cells was much slower than NT precursor synthesis, b) that NT(3-13) was a major product and NT a minor one, and c) that NT precursor and its products were stored within secretory vesicles.