The generation of a chemotactic factor by guinea pig alveolar macrophages after phagocytosis of heat-killed Staphylococcus aureus was studied. These studies demonstrated that after phagocytosis, alveolar macrophages secrete a small molecular weight (less than 5,000 daltons) chemotactic factor that preferentially attracts neutrophils. The chemotactic factor can be generated in vivo and in vitro, and its chemotactic activity can be detected both in vitro by a chemotactic assay and in vivo by an increase in the absolute number of neutrophils in bronchoalveolar lavage fluid and in lung tissue histologic specimens. Generation of the chemotactic factor was inhibited by 20 microgram of actinomycin D per ml and by 10 microgram of cycloheximide per ml. The factors were stable after incubation at 56 degrees C for 45 min, but not after incubation at 100 degreeg C for 10 min. These studies show that alveolar macrophages can serve as an initiator of pulmonary inflammatory responses by secreting a potent mediator of neutrophil locomotion.