Fibronectin (FN), a glycoprotein present in the plasma and the extracellular matrix, has been shown to enhance adherence-related functions of polymorphonuclear leukocytes (PMNs). In this study we investigated the effects of FN on the activation of human PMNs in suspension by soluble stimuli, as determined by the generation of superoxide radicals (respiratory burst). FN (up to 100 micrograms/ml) did not directly stimulate the PMN respiratory burst assessed using a sensitive assay, luminol-dependent chemiluminescence (CL). Low FN concentrations (Up to 25 micrograms/ml) caused a dose-dependent enhancement of the CL induced by two chemoattractants. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and platelet-activating factor (Paf), and also by phorbol myristate acetate (PMA), a known protein kinase C activator. Higher FN concentrations were less effective. The potentiation involved both initial rate and total CL responses and was more active on extracellular than intracellular generation of oxygen radicals. FN potentiation persisted after cell washing and was abolished by treatment of FN with trypsin. Measurement of the respiratory burst using the cytochrome c reduction assay confirmed that FN enhanced both the initial rate and total amount of superoxide anion generated by FMLP-stimulated PMNs. These data indicate that FN facilitates the respiratory burst of chemoattractant-stimulated PMNs and suggest that FN can prepare PMNs in suspension for amplified biological functions induced by soluble inflammatory stimuli.