Although isolated islets from older rats store ample insulin, they do not respond as efficiently as do islets from young animals to a glucose or leucine challenge. The current study was conducted to determine if this decline in insulin secretion is associated with abnormalities in islet content or conversion of proinsulin, or in the mobilization of insulin that is newly synthesized. Isolated islets from 2-, 12-, and 18-mo-old rats were assayed for total islet proinsulin and insulin content, and after stimulation with glucose the incubation medium was assayed for secreted proinsulin and insulin. In addition, islets from 2- and 12-mo-old donors were pulse-labeled with 3H-leucine, after which the temporal conversion and pattern of release of newly synthesized hormone was compared. The results show that the ratio of proinsulin to insulin declines progressively with age for both the stored and secreted hormone. Incorporation of 3H-leucine into proinsulin during glucose stimulation also decreases with age. However, the onset, time course, and endpoint of the conversion of this labeled proinsulin to insulin did not change with age. Although the fractional secretion rate of newly synthesized hormone (whether proinsulin or insulin) is similar in 2- and 12-mo-old rats, it exceeds that of the unlabeled, or stored, insulin. When the wave of labeled protein enters the secretory pool it apparently equilibrates with a portion of the insulin pool, which is more rapidly secreted than the total pool of insulin. We considered this the glucose responsive pool, and estimate that this pool is actually smaller in islets from 12- as compared with 2-mo-old rats. The reduction in size of this responsive compartment of insulin in the older rats may, in part, explain the decreased rates of stimulated insulin release that OCCUr with age.