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We investigated, in three beta cell lines (INS-1E, RIN-5AH, betaTC3) and in human and rodent primary beta cells, the storage and release of chromogranin B, a secretory protein expressed in beta cells and postulated to play an autocrine role. We asked whether chromogranin B is stored together with and discharged in constant ratio to insulin upon various stimuli.The intracellular distribution of insulin and chromogranin B was revealed by immunofluorescence followed by three-dimensional image reconstruction and by immunoelectron microscopy; their stimulated discharge was measured by ELISA and immunoblot analysis of homogenates and incubation media.Insulin and chromogranin B, co-localised in the Golgi complex/trans-Golgi network, appeared largely segregated from each other in the secretory granule compartment. In INS-1E cells, the percentage of granules positive only for insulin or chromogranin B and of those positive for both was 66, 7 and 27%, respectively. In resting cells, both insulin and chromogranin B were concentrated in the granule cores; upon stimulation, chromogranin B (but not insulin) was largely redistributed to the core periphery and the surrounding halo. Strong stimulation with a secretagogue mixture induced parallel release of insulin and chromogranin B, whereas with 3-isobutyl-1-methylxantine and forskolin ± high glucose release of chromogranin B predominated. Weak, Ca2+-dependent stimulation with ionomycin or carbachol induced exclusive release of chromogranin B, suggesting a higher Ca2+ sensitivity of the specific granules.The unexpected complexity of the beta cell granule population in terms of heterogeneity, molecular plasticity and the differential discharge, could play an important role in physiological control of insulin release and possibly also in beta cell pathology.