Insulin and proinsulin syntheses from leucine-H-3 were studied in glucose-stimulated perifused rat islets, with particular attention being placed on the contribution of de novo synthesis to the characteristic second phase of insulin release. At fifty to sixty minutes, when the second phase had reached approximately steady state, most of the islet radioactivity was still in the proinsulin fraction; newly synthesized insulin during this interval represented less than 0.5 per cent of the total insulin secreted. By 110 to 120 minutes, this value had increased, but to less than 17.5 per cent. The half time of conversion of proinsulin to insulin was approximately sixty minutes. At either fifty to sixty or 110 to 120 minutes, proinsulin was less than 5 per cent of the total hormone secreted. The ratio of specific activities for secreted to islet insulin was less than unity at sixty minutes and exceeded unity at 120 minutes; the comparable ratios for proinsulin were somewhat above unity at both times. Results suggest newly synthesized insulin is not responsible for the second phase of insulin release. In addition, storage of insulin is nonhomogeneous with preferential release of the older nonlabeled hormone occurring during the first hour of glucose stimulation.