This study has been planned to investigate some aspects of the interaction between acetylsalicylic acid (ASA) and tolbutamide on insulin secretion. In healthy subjects, oral administration of 3.2 g daily of ASA for 3 days significantly enhanced a) basal insulin levels (p less than 0.01), b) arginine-stimulated insulin secretion (25 g i.v. over 30 min) (p less than 0.01) and c) tolbutamide-stimulated insulin secretion (1 g or 0.25 g i.v. as a bolus) (areas under curves: p less than 0.02). Corresponding decreases in glycemia were observed. Tolbutamide binding to serum proteins was significantly reduced after ASA treatment (p less than 0.02). We conclude that, in case of tolbutamide test, interferences between ASA and tolbutamide on insulin secretion might be dependent, at least in part, on enhancement of free-tolbutamide percentage in plasma and not only on a direct or synergic action of ASA on pancreatic B-cell. Therefore, acute stimulation of insulin secretion by tolbutamide appears not to be completely comparable to other traditional stimuli, when ASA effects are studied.