Human milk neutrophils and macrophages were examined by flow cytometry to determine whether they displayed phenotypic markers of activation. The markers were CD11b and L-selectin, which are increased or shed, respectively, from the surface of activated neutrophils. Phenotypic features of milk neutrophils and macrophages were similar to blood neutrophils stimulated with fMLP: plasma membrane expression of CD11b was increased and L-selectin was decreased. After blood neutrophils were incubated in acellular milk, their expression of CD11b increased and L-selectin decreased. The activation was not affected by trypsin but was significantly decreased by treating acellular milk with chloroform or ether. Sedimentation studies suggested that particulate fractions of milk were active. Further, the activation was partly blocked by treating target blood neutrophils with cytochalasin B. Thus, human milk neutrophils are activated, and the activation may be due partly to phagocytosis of membranous structures in milk.