It is shown how serial block-face electron microscopy (SBEM) of insulin-secreting β-cells in wild-type mouse pancreatic islets of Langerhans can be used to determine maturation times of secretory granules. Although SBEM captures the β-cell structure at a snapshot in time, the observed ultrastructure can be considered representative of a dynamic equilibrium state of the cells since the pancreatic islets are maintained in culture in approximate homeostasis. It was found that 7.2 ± 1.2% (±st. dev.) of the β-cell volume is composed of secretory granule dense-cores exhibiting angular shapes surrounded by wide (typically ≳100 nm) electron-lucent halos. These organelles are identified as mature granules that store insulin for regulated release through the plasma membrane, with a release time of 96 ± 12 h, as previously obtained from pulsed 35S-radiolabeling of cysteine and methionine. Analysis of β-cell 3D volumes reveals a subpopulation of secretory organelles without electron-lucent halos, identified as immature secretory granules. Another subpopulation of secretory granules is found with thin (typically ≲30 nm) electron-lucent halos, which are attributed to immature granules that are transforming from proinsulin to insulin by action of prohormone convertases. From the volume ratio of proinsulin in the immature granules to insulin in the mature granules, we estimate that the newly formed immature granules remain in morphologically-defined immature states for an average time of 135 ± 14 min, and the immature transforming granules for an average time of 130 ± 17 min.