The dynamics of insulin release by pieces of fetal rat pancreas from 17.5 to 21.5 days of gestation was measured in an in vitro perifusion system. Interactions between glucose, theophylline, and a mixture of 12 amino acids at physiologic concentration (mix. A.A.) were studied. On day 17.5, 13.9 mM glucose induced only a small (10 min) early phase of insulin release. The late phase of insulin secretion appeared on day 18.5 and gradually increased as the gestation proceeded. The mix. A.A. (9 mM) or theophylline (5 mM) potentiated the two phases of insulin release induced by 13.9 mM glucose from days 18.5 to 21.5 of gestation. On day 21.5, the combination of theophylline and mix. A.A. at 2.2 mM glucose stimulated insulin release by fetal pancreas. The dose-dependent curves of the early phase of insulin release, due to glucose or glucose and mix. A.A. (9 mM), showed half maximal responses in the term fetal pancreas at glucose concentrations of 7.2 and 4 mM, respectively. The values were 7.9 and 5.9 mM for the late phase. The results indicate that the mechanisms controlling biphasic insulin release develop during the late fetal life in the rat. Transition from the fetal to adult type of insulin secretion more likely parallels quantitative, rather than qualitative, changes within the B cells.