Rapid separation of avian heterophils from anticoagulated whole blood was achieved using Ficoll-Hypaque discontinuous gradients. An average of 14.4% of blood heterophils was harvested with a mean purity exceeding 99%. Heterophil viability, as determined by trypan blue dye exclusion, averaged 99.8%. The integrity of isolated heterophils was evaluated by cytochemical staining and ultrastructural examination. Cytochemical staining reactions of heterophils in whole blood and of isolated cell suspensions were similar. No ultrastructural abnormalities were observed. Using this procedure, viable intact heterophils were rapidly isolated from blood with an acceptable cell yield and purity for cell function studies.