There is growing information about the heterogeneity of pancreatic β-cells and how it relates to insulin secretion. This study used the approach of flow cytometry to sort and analyze β-cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter. Three populations of β-cells with differing GFP brightness could be identified, which were classified as GFP-low, GFP-medium, and GFP-bright. The GFP-medium population comprised about 70% of the total. The GFP-low population had less insulin secretion as determined by the reverse hemolytic plaque assay and reduced insulin gene expression. Additionally, all three subpopulations of β-cells were found in mice of varying ages (embryonic d 15.5 and postnatal wk 1–9). The three populations from the youngest had larger cells (forward scatter) and less granularity (side scatter) than those from the adults. This approach opens up new ways to advance knowledge about β-cell heterogeneity.