This study evaluated the role played by cholecystokinin (CCK) receptors' occupation in the control of somatostatin (SS) secretion in RIN-14B cells.The presence of the CCK receptors 1 and 2 was confirmed by immunofluorescence, and SS secretion was evaluated by enzyme-linked immunosorbent assay.By immunofluorescence, 95% of the cell population was composed of SS cells bearing both CCK-R subtypes with 5% of beta cells (data not shown). Cerulein (Cae), a CCK-1R agonist, and pentagastrin, a CCK-2R agonist, dose-dependently increased SS release, 3-fold at 1 mumol/L Cae, 2.5-fold at 10 mumol/L pentagastrin, with occupation of both CCKRs confirmed by L-364,178 and L-365,260 inhibition of CCK receptors 1 and 2. The occupation of high-affinity CCK-1R by Cae was confirmed on SS release with JMV-180, a high-affinity CCK-1R agonist, and absence of SS release inhibition at high Cae concentration occupying the low-affinity CCK-1R. These cells release more than 60% of their SS content by constitutive secretion, confirmed by cycloheximide and brefeldin inhibiting SS synthesis and intracellular trafficking, respectively.Both CCKR subtypes occupy RIN-14B cells and initiate SS secretion through constitutive secretion controlled at SS synthesis level. Somatostatin secretion via the CCK-1R occupation mobilizes its high-affinity sites.