Manganese (Mn) is a calcium (Ca) analog that has long been used as a magnetic resonance imaging (MRI) contrast agent for investigating cardiac tissue functionality, for brain mapping and for neuronal tract tracing studies. Recently, we have extended its use to investigate pancreatic β-cells and showed that, in the presence of MnCl2, glucose-activated pancreatic islets yield significant signal enhancement in T1-weigheted MR images. In this study, we exploited for the first time the unique capabilities of X-ray fluorescence microscopy (XFM) to both visualize and quantify the metal in pancreatic β-cells at cellular and subcellular levels. MIN-6 insulinoma cells grown in standard tissue culture conditions had only a trace amount of Mn, 1.14 ± 0.03 × 10−11 µg/µm2, homogenously distributed across the cell. Exposure to 2 m m glucose and 50 µ m MnCl2 for 20 min resulted in nonglucose-dependent Mn uptake and the overall cell concentration increased to 8.99 ± 2.69 × 10−11 µg/µm2. When cells were activated by incubation in 16 m m glucose in the presence of 50 µ m MnCl2, a significant increase in cytoplasmic Mn was measured, reaching 2.57 ± 1.34 × 10−10 µg/µm2. A further rise in intracellular concentration was measured following KCl-induced depolarization, with concentrations totaling 1.25 ± 0.33 × 10−9 and 4.02 ± 0.71 × 10−10 µg/µm2 in the cytoplasm and nuclei, respectively. In both activated conditions Mn was prevalent in the cytoplasm and localized primarily in a perinuclear region, possibly corresponding to the Golgi apparatus and involving the secretory pathway. These data are consistent with our previous MRI findings, confirming that Mn can be used as a functional imaging reporter of pancreatic β-cell activation and also provide a basis for understanding how subcellular localization of Mn will impact MRI contrast. Copyright © 2011 John Wiley & Sons, Ltd.