Amidated and non-amidated progastrin-derived peptides have distinct biological activities that are mediated by a range of receptor subtypes. The objective was to determine the nature of the stored and secreted progastrin-derived peptides and to investigate whether progastrin release is regulated by gastric acidity. Using an antiserum directed to the C-terminus of progastrin for identification and to monitor purification, C-terminal flanking peptides (CTFP) of progastrin (prog_76-83, prog_77-83 and prog_78-83 in approximately equivalent amounts) were isolated and identified from extracts of sheep antrum using ion exchange, HPLC and mass spectrometry. Only trace amounts of full length progastrin were present. Progastrin CTFP was the predominant progastrin-derived peptide in the antrum (Progastrin CTFP/Gamide = 3). Similarly, progastrin CTFP was the major circulating form in the antral (CTFP 710 ± 62 pmol/liter, gastrin amide 211 ± 35 pmol/liter) and jugular veins (CTFP 308 ± 16 pmol/liter, gastrin amide 32 ± 3 pmol/liter). Alteration of gastric acidity in sheep by intravenous infusion of a H/K ATPase inhibitor or somatostatin, or by intragastric infusion of HCl, demonstrated that the CTFP concentrations changed, although to a lesser extent than the changes in circulating gastrin amide. We conclude that the CTFP of progastrin is the major stored and circulating species of the gastrin gene and that it is secreted in a regulated fashion rather than constitutively. As full length progastrin is bioactive but is only a minor antral and secreted form, determination of the biological activity of the C-terminal flanking peptides will be important for a complete understanding of gastrin endocrinology.