Acute insulin secretion from stimulated pancreatic beta-cells is derived from the intracellular pool of insulin secretory granules wherein insulin is packaged in a highly concentrated (and in some species, crystalline) state. Here we review experimental work, principally from our laboratory, on the question of biogenesis of mature secretory granules within the broader context of intracellular protein trafficking. Events occurring in the lumen of organelles at various stages of intracellular transport within the secretory pathway and events at the limiting membrane of newly forming secretory granules each contribute to formation of the insulin storage compartment comprising the readily releasable pool.