Pituitary cells from 14-day-old rats were separated by unit gravity velocity sedimentation, and a highly enriched population of gonadotrophs was established in reaggregate cell culture in serum-free and serum albumin-free defined culture media. The medium conditioned by these aggregates was concentrated, ultrafiltrated, and the concentrated substances consecutively separated by two reversed-phase HPLC steps, a gel filtration step and an additional reversed-phase purification step. Two peptides could be isolated that displayed an N-terminal amino acid sequence identical to fragments of rat presecretogranin II: one was cleaved at the dibasic amino acid residues K182-R183 and the other at the dibasic amino acid residues K569-R570. The latter peptide was completely purified and separated into three variants with the same N-terminal amino acid sequence. From an analysis by electrospray ionization mass spectrometry, it was deduced that the three forms extended up to amino acid residue 612 (A), which is, in presecretogranin II, also flanked by two basic amino acids (K613R614). Two variant forms had a molecular mass that was 17 Da higher. The present data support the hypothesis that secretogranin II is a precursor of putative regulatory peptides.