Refractoriness of the pancreatic β-cell to glucose stimulation plays a role in the secretory defect of NIDDM, but the mechanisms underlying this refractoriness are not clear. The purpose of this study was to determine whether the HIT-T15 pancreatic β-cell line can be used as an investigative model for refractoriness of glucose-induced insulin secretion and, if so, whether the mechanism for this refractoriness involves alteration in stimulus-secretion coupling (desensitization) or results from exhaustion Of insulin stores. In perfusion experiments, acute insulin responses (AIRs) in HIT-T15 cells progressively diminished during consecutive 5-min glucose (11.1 mmol/l) pulses (G) given every 20 min (G1 = 9.2 ± 1.3, G2 = 4.1 ± 1.0, G3 = 2.7 0.7, G4 = 2.5 ± 1.1 μU/ml). To determine whether this refractoriness for glucose extended to the potentiating effects of glucose on nonglucose secretagogues, cells were challenged with arginine after desensitization with glucose. In HIT-T15 cells, the response to the arginine pulse (16.7 ± 5.2 μU/ml) after three glucose pulses was significantly less (P < 0.01) than the response to a control arginine pulse (29.6 ± 12.1 μU/ml) preceded by an infusion of buffer in the absence of glucose pulses. Variable rest periods after desensitization allowed recovery of the AIR in HITT15 cells; responses 30, 60, 90, and 120 min after the desensitization procedure increased in a stepwise fashion (3.8 ± 2.7, 4.5 ± 2.7, 7.8 ± 5.2, and 9.7 ± 5.3 μU/ml, respectively). To differentiate desensitization from exhaustion of insulin stores, studies were performed in the presence of epinephrine, a potent inhibitor of insulin secretion. In HIT-T15 cells, after three pulses of glucose during the epinephrine infusion, epinephrine was discontinued and the insulin response to a fourth pulse was assessed. The G4 AIR (1.9 ± 0.6 U/ml) was markedly less than a control G4AIR (5.4 1.2 μU/ml) that followed an epinephrine infusion alone with no concurrent glucose pulses. β-cell refractoriness was also induced in the HIT-T15 cell using 45- min steady-state infusions of glucose. Cells were exposed to a 45-min infusion of either 3.7 or 11.1 mmol/l glucose, rested for 20 min in the absence of glucose, and then challenged with a 5-min, 11.1 mmol/l glucose pulse. In both cases, the AIR to the 5-min pulse (10.2 ± 5.1 and 2.9 ± 1.4 μU/ml after the 3.7 and 11.1 mmol/l infusion, respectively) was lower than the AIR to a control pulse (27.4 ± 5.9 μU/ml) not preceded by glucose infusion. These studies demonstrated that the HIT-T15 cell line is an appropriate model for studying mechanisms of β-cell refractoriness to glucose signaling. The short-term intensive glucose stimulation paradigms used in our studies induced an abnormality in insulin secretion but not β- cell exhaustion.