Fibronectin is capable of activating macrophages for enhanced nonopsonic phagocytosis of Pseudomonas aeruginosa grown in vivo in rats or mice or in vitro on nutrient agar plates. In this study it was determined that while fibronectin was able to significantly increase phagocytosis of organisms grown in static broth, uptake of agitated bacteria could not be promoted. Agitated P. aeruginosa cultures were proven to lack surface pili expression, as assessed by electron microscopic studies. A pilus-deficient pilA::Tn501 mutant of P. aeruginosa PAO was constructed by gene replacement techniques. Phagocytosis of this mutant could not be enhanced by fibronectin regardless of growth conditions. Furthermore, 60 micrograms of exogenously added Pseudomonas pili per ml was capable of abrogating the enhanced phagocytosis of the wild-type strain observed with fibronectin-stimulated macrophages. It is concluded that Pseudomonas pili were the bacterial ligands required for attachment to fibronectin-stimulated macrophages in the initial stages of nonopsonic phagocytosis.