Efficient polymorphonuclear neutrophil (PMN) migration depends on specific interactions between PMNs, endothelial cells, and extracellular matrix (ECM) proteins. We investigated the relationship between PMN migration and the ECM molecule fibronectin (FN). We used an in vitro migration assay system to show that human PMNs migrated across an FN-coated filter barrier toward a formyl-Met-Leu-Phe (fMLP) chemoattractant gradient in greater numbers than across (uncoated) bare fitters. In 1 h of fMLP stimulation, 69 +/- 6% of the PMNs had migrated across the FN-coated filters, whereas 46 +/- 5% of PMNs migrated across bare filters. This effect was specific to FN; coating the filters with the ECM protein vitronectin did not enhance migration. Monoclonal antibodies against FN or against the alpha5 or beta1 integrin subunits of the FN receptor inhibited the enhanced PMN migration response across FN-coated filters. These findings indicate that the extracellular matrix protein FN enhances PMN migration and that this response is mediated by the alpha5beta1 FN receptor.