Cholecystokinin (CCK) is a gut hormone that acts via two receptors. The CCKA-receptor requires the tyrosyl residue in the C-terminal bioactive site of CCK to be O-sulfated, whereas, the CCKB-receptor binds irrespective of sulfation. Consequently, unsulfated CCK peptides - if present - may constitute a hormone system that acts only through the CCKB-receptor. Therefore, we have now examined whether, CCK peptides occur in nonsulfated form in the small intestine of pigs and rats. The concentrations of sulfated and nonsulfated CCK were measured by RIAs, one specific for sulfated CCKs and a new two-step assay specific for nonsulfated CCK. For further characterization, the intestinal extracts were subjected to size- and ion exchange-chromatography. The intestinal concentrations of sulfated and nonsulfated CCK were highest in the duodenum and the proximal part of jejunum both in the pig and the rat. The porcine duodenal mucosa contained 193±84pmol/g sulfated CCK and 31±10pmol/g nonsulfated CCK, and the upper rat intestine 70±19pmol/g and 8±2pmol/g, respectively. The degree of sulfation correlated with the endoproteolytic proCCK processing. Thus, 38% of porcine CCK-58 was unsulfated, whereas, only 12% of CCK-8 was unsulfated. The results show that a substantial part of intestinal CCK peptides in rats and pigs are not sulfated, and that the longer peptides (CCK-58 and CCK-33) are less sulfated than the shorter (CCK-22 and CCK-8). Hence, the results demonstrate that proCCK in the gut is processed both to sulfated and unsulfated α-amidated peptides of which the latter are assumed to act via the CCKB-receptor.