A method is presented to separate turkey heterophils from anticoagulated whole blood using two-step Ficoll-Hypaque discontinuous gradients and ammonium chloride lysis of contaminating erythrocytes. Heterophils can be isolated from multiple blood samples within 3 to 4 hours. Using this technique, 66.4 +- 18.4% (mean +- standard deviation) of blood heterophils were harvested. Final cell isolates averaged 96.0 +- 2.9% heterophils with few contaminating eosinophils (2.5 +- 2.3%) or basophils (1.6 +- 1.8%). Cell viability, as determined by trypan blue dye exclusion, was 98.0 +- 1.4%.