The aim of this study was to evaluate the effects of long term in vitro exposure of human pancreatic islets to different secretagogues on their subsequent secretory activity. Therefore, groups of 100 islets were cultured for 48 h in standard tissue culture medium (CMRL 1066) in the presence of 1 of the following: 5.5 mmol/L glucose, 16.7 mmol/L glucose, 5.5 mmol/L glucose plus 10 mmol/L L-arginine, or 5.5 mmol/L glucose plus 100 mumol/L tolbutamide. Insulin levels in the culture medium declined with time under all culture conditions. Islets were then perifused and acutely stimulated with glucose (16.7 mmol/L), L-arginine (10 mmol/L), and tolbutamide (100 mumol/L). Islets cultured in 16.7 mmol/L glucose showed no response to 16.7 mmol/L glucose [net area under the curve (delta AUC), 11% of control], and a reduced response to acute tolbutamide (delta AUC, 35% of control), but responded to L-arginine (delta AUC, 75% of control). Islets cultured in the presence of 10 mmol/L L-arginine had reduced responses to glucose (delta AUC, 11% of control) and tolbutamide (delta AUC, 27% of control), but responded to L-arginine (delta AUC, 75% of control). Islets cultured in tolbutamide did not respond to tolbutamide (delta AUC, 14% of control) and showed a reduced responses to acute glucose (delta AUC, 36% of control) and L-arginine (delta AUC, 24% of control). In a second set of experiments, islets cultured in 5.5 or 16.7 mmol/L glucose showed an insulin response to a supramaximal glucose stimulation (30 mmol/L glucose plus 0.5 mmol/L isobutylmethylxanthine) that was not statistically different. Similarly, islets that were cultured in the presence of 100 mumol/L tolbutamide still responded to 1 mmol/L tolbutamide. In conclusion, all stimuli evaluated in this study, chronically applied, reduced the insulin response to further acute stimulations. The different patterns of unresponsiveness observed together with the finding of a preserved insulin content in the islets after perifusions and a maintained capability to release insulin in response to supramaximal stimulations suggest that after chronic exposure to different stimuli, human islets become selectively desensitized to the same stimuli given acutely and do not become exhausted.