Eosinophils contain many preformed cytokines and chemokines, which are stored in specific granules along with cationic granule proteins. Mobilization and release of these granule contents can be selective and mediated by vesicular transport. We have developed a sensitive method to detect and quantitate eosinophil vesicular transport-mediated release of specific eosinophil proteins. Our EliCell assay is based on microscopic observations of individual viable eosinophils embedded in an agarose matrix that contains immobilized antibody to the protein of interest. Following stimulation of eosinophils, released protein is bound by the capture antibody at its site of release and is detected by a fluorochrome-conjugated detection antibody. We have validated this assay by evaluating interferon-gamma-induced release of RANTES from eosinophils. Extracellularly released RANTES was visualized as focal immunoflourescent staining and was quantitated by scoring the numbers of eosinophils releasing RANTES and by measuring the fluorescent intensity over individual eosinophils. In comparison with ELISA assays of RANTES released into supernatant fluids by interferon-gamma-stimulated eosinophils, EliCell assays were more sensitive enabling detection of RANTES release at earlier times and at lower levels of interferon-gamma stimulation. The EliCell assay provides a sensitive method to study the regulated release of eosinophil-derived cytokines, chemokines and other granule proteins.