The dynamics of insulin release in response to relatively long infusions of glucose were studied in the isolated perfused rat pancreas. Insulin secretion was determined by immunochemical assay of the total portal vein effluent. Histological examination of the perfused pancreases and measurement of oxygen consumption by these tissues indicated that optimal physiological conditions were used. It was observed that, when glucose was infused for a period of approximately 1 hr into a perfused pancreas, there appeared 2 distinctly different phases of insulin release. There was an early, or rapid, release of insulin which subsided within approximately 2 min, followed by a late, or slow release phase which continually increased in rate until termination of the glucose infusion. The contribution of newly synthesized insulin to either phase was determined by comparing the insulin release by normal control preparations to that by preparations which were treated with puromycin. Incorporation of L-valine-¹⁴C was used as the means of detecting the effect of puromycin onpancreatic protein synthesis. It was found that the rapid response was unaffected by the puromycin treatment, but the slow response was definitely depressed in the puromycin-treated preparations, indicating that newly synthesized insulin plays an integral part in this second insulin response. In contrast, tolbutamide caused the rapid response, but no significant slow response was detected, suggesting that this agent has no effect on insulinogenesis and/or the mechanism which gives rise to the second phase. In addition, the total amount of insulin released during both the early and late phase was proportional to the amount of calcium present in the perfusing medium up to a concentration of approximately 4 mEq/1. At calcium levels greater than this, no further increase in the total amount of insulin released was observed. Calcium had little effect on ¹⁴C-labeled amino acid incorporation into pancreatic protein, indicating that this cation is required for the normal insulin secretory process rather than affecting insulinogenesis. (Endocrinology83: 572, 1968)