Highly purified eosinophils can be isolated from peripheral blood by negative selection using anti-CD16 immunomagnetic beads. The basic protocol describes a sequential fractionation of peripheral blood in which eosinophils are separated first from mononuclear cells and then from CD16(+) neutrophils. This technique is easy to use, fast, and highly reproducible. A support protocol describes a staining method that can be used to evaluate the purity of eosinphils.