Subcellular fractionation has been an important tool in investigating human eosinophil structure and function, including localizing of cytokine/chemokines within granules, investigating granule protein translocation and intracellular transport during eosinophil secretion, and studying secretory mechanisms of granules. The resolution of organelles obtained by subcellular fractionation was improved considerably after the introduction of nonionic iodinated density-gradient metrizamide and Nycodenz media that, unlike sucrose, exhibit relatively low tonicity throughout the gradient. However, the structure and membrane preservation of isolated organelles were still compromised due to the lack of gradient isoosmolarity. This paper describes a detailed protocol of subcellular fractionation of nitrogen cavitated eosinophils on an isoosmotic iodinated density gradient (iodixanol - OptiPrep) and the isolation of well preserved and functional membrane-bound specific granules.