The dynamics of insulin release were investigated in vitro in order to determine the regulatory processes governing its biphasic shape. When subjected to a square wave glucose stimulation, the isolated perfused rat pancreas responded with typical biphasic insulin release. Both the duration of the nadir between the two phases and the slope of recovery of insulin release during second-phase secretion exhibited glucose dose dependency. Successive 40-min stimuli with glucose (8.3 and 16.7 mM), separated by a 20-min rest period, resulted in 2.6- 3.3-fold potentiation of the early phase insulin release rate, previously described as glucose-primed time-dependent potentiation (TDP) of insulin secretion. A linear relationship (r = 0.89, P less than 0.001) was observed between the degree of TDP and the slope of second-phase insulin release. Successive short stimuli with glucose (5-10 min long, 5-10 min apart; 6.9, 8.3, and 16.7 mM) resulted in the inhibition of the response to the second stimulus; this effect was termed time-dependent inhibition (TDI) of insulin release. Arginine also induced TDI; this was completely overcome by synergistic interaction with glucose (8.3 mM). The glucose-arginine interaction was utilized to demonstrate that the interphasic nadir of insulin release was the expression of TDI. Thus, introduction of an arginine stimulus during the nadir in glucose-induced insulin release abolished the silent phase, the secretion rate reaching the level expected for the combined glucose-arginine stimulus. However, the continued presence of TDI could be demonstrated by removal of the arginine stimulus, at which time, despite ongoing glucose stimulation, insulin secretion was markedly inhibited. These observations support the concept that the biphasic dynamics of insulin release is the net expression of three regulatory processes: 1) the acute stimulus-secretion coupling system, best observed as the immediate, first-phase response to a stimulus; 2) TDI of insulin release, a relatively rapid signal responsible for the silent period; and 3) TDP of insulin release, a slow rising signal responsible for recovery from the silent phase, building up the second-phase of secretion.