The multistep model of leukocyte adhesion reveals that selectins mediate rolling interactions and that integrins mediate firm adhesion processes. In this study, the interaction between eosinophils and TNF-alpha-activated HUVEC (second or third passage) was studied under flow conditions (0.8 and 3.2 dynes/cm2). Especially the role of alpha 4 integrins on eosinophils and E-selectin on HUVEC was studied. Inhibition of the integrin alpha 4 chain on eosinophils reduced the number of firmly adhered resting eosinophils to TNF-alpha-stimulated endothelium by 43% whereas the percentage rolling cells increased 2.2-fold compared with untreated control eosinophils. Blocking of E-selectin on the endothelium reduced the number of adherent eosinophils by only 23% and 16%. In this situation, however, hardly any rolling adhesion was observed, and the few rolling cells showed a low rolling velocity. Blocking both alpha 4 integrin on eosinophils and E-selectin on HUVEC reduced the number of adhered eosinophils by 95%. P-selectin did not significantly participate in eosinophil adhesion to TNF-alpha-activated HUVEC. Inhibition of both alpha 4 integrins and beta 2 integrins on eosinophils resulted in a reduction of adhered cells by 65% and a 3-fold increase in percentage rolling cells. Taken together, these results clearly show that resting eosinophils preferentially use constitutively active alpha 4 integrins (alpha 4 beta 1, alpha 4 beta 7) for the first attachment to TNF-alpha-activated HUVEC. In addition, alpha 4 integrins and E-selectin work synergistically in eosinophil adherence to TNF-alpha-activated HUVEC. Although E-selectin is important for eosinophil rolling under these conditions, P-selectin plays only a minor role.