Background: Eosinophils that have bound to extracellular matrix proteins, such as the connecting segment 1 (CS-1) region of fibronectin, need to deadhere before undergoing chemotaxis through the extracellular matrix. Objective: We have investigated whether eotaxin can regulate the strength of eosinophil adhesion to the CS-1 region of fibronectin. Methods: We have used a micropipette single-cell adhesion assay to determine the force of eosinophil adhesion to the CS-1 region of fibronectin. Results: Eosinophils bound to CS-1 with high avidity, and this binding could be inhibited with neutralizing antibodies to ⍺4 integrins expressed by eosinophils or with neutralizing antibodies to CS-1. Eosinophils incubated in the presence of eotaxin demonstrated a transient increase in the force of eosinophil adhesion to CS-1, which was followed by a more sustained reduction in the force of eosinophil adhesion to CS-1, as assessed in the micropipette single-cell adhesion assay. This decreased binding of eosinophils to CS-1 was not due to alterations in very late antigen 4 (VLA-4) receptor number, as assessed with FACS analysis, or alterations in VLA-4 receptor distribution, as assessed with immunofluorescence microscopy. Conclusions: These studies suggest that eotaxin can cause a transient increase followed by a more sustained reduction in the functional force of VLA-4 adhesion to CS-1 and thus promote deadhesion of CS-1 adherent eosinophils in the extracellular matrix. (J Allergy Clin Immunol 2000;106:933-40.)