Aging in men and rodents is associated with a marked decline in glucose stimulated insulin secretion by pancreatic beta cells (B cells). Secreted insulin is the end result of a series of steps along the biosynthetic protein-secretion pathway, including insulin gene transcription, processing of transcripts to preproinsulin mRNA, translation of mRNA, segregation and processing of newly made proinsulin in secretory vesicles, proinsulin to insulin conversion, transport of vesicles to the plasma membrane, and exocytosis. We have examined the influence of age at three stages along this pathway: preproinsulin mRNA levels, proinsulin synthesis, and secretion of newly made and preformed insulin, using Fischer rats, a widely studied rodent model of aging. Pancreatic weights and total insulin contents, islet sizes, and mean insulin content per islet were the same in young adult (4-5 mo) and senescent (21-22 mo) animals. There was no effect of age on preproinsulin mRNA levels in whole pancreata of fed animals, or in isolated islets cultured for 16 h in 5.5 mM glucose. Proinsulin biosynthesis and the secretion of newly made insulin were compared in isolated islets preincubated in 5.5 mM glucose. After a pulse label at 16.7 mM glucose, proinsulin synthesis, assayed by immunoprecipitation, was decreased 16% in 7 mo islets and 39% in 21-22 mo islets, compared with 4-5 mo islets, though total protein synthesis was not reduced. When chased at 2.8 mM glucose, 4-5 month and 21-22 mo islets showed no difference in release of preformed or newly made insulin. When chased at 16.7 mM glucose, there was a significant decrease in the secretion of newly made insulin in the old islets compared with the young islets. There was preferential release of newly made insulin over preformed insulin in both young and old islets. However, since secretion of preformed insulin was decreased much more than secretion of newly made insulin in senescent islets, these displayed a two- to threefold increase in the proportion of newly made insulin relative to total immunoreactive insulin released compared with young adult islets. The differential effects of aging on these steps in the insulin synthesis-secretion pathway may be due to varying impairments in signals transducing the glucose stimulus into the wide range of B cell responses to glucose.