The existence of morphologically distinct populations of islets in the pancreas was described over 60 years ago. Unfortunately, little attention has been paid to possible functional differences between islet subpopulations until recently. We demonstrated that one population, the small islets, were superior to large islets in a number of functional aspects. However, that work did not determine whether these differences were inherent, or whether they arose because of the challenge of isolation procedures. Nor, were there data to explain the differences in insulin secretion. We utilized immunohistochemistry, immunofluorescence, ELISA, and transmission electron microscopy to compare the unique characteristics found in isolated rat islet populations in situ and after isolation. Insulin secretion of small isolated islets was significantly higher compared to large islets, which correlated with higher insulin content/area in small islets (in situ), a higher density of insulin secretory granules, and greater insulin content/volume in isolated islets. Specifically, the core b-cells of the large islets contained less insulin/cell with a lower insulin granule density than peripheral b-cells. When insulin secretion was normalized for total insulin content, large and small islets released the same percentage of total insulin. Small islets had a higher density of cells/area than large islets in vitro and in situ. The data provide a possible explanation for the inferior insulin secretion from large islets, as they have a lower total cell density and the b-cells of the core contain less insulin/cell.