—Atherosclerotic lesion development seems to be inflammatory in nature and involves the recruitment of monocytes to the vessel wall. In this study, we investigated the role of vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (FN) connecting segment-1 containing the amino acid sequence ILDV as functional ligands for α₄β₁ integrin (VLA-4) in monocyte rolling and adherence to early atherosclerotic lesions. Carotid arteries of apolipoprotein E–deficient mice were isolated and perfused with monocytes or U937 cells. Cell adhesion was reduced 95±4% by monoclonal antibodies HP1/2 and HP2/1, which block VLA-4 binding to both VCAM-1 and FN connecting segment-1. mAb HP1/3 preferentially blocked interaction of VLA-4 with FN but not VCAM-1 and decreased adhesion by 30±8%. In contrast, blocking VCAM-1 by perfusing the isolated carotid artery with mAb MK-2.7 reduced adhesion by 75±12%. Mononuclear cell adhesion to the early atherosclerotic endothelium was inhibited by 68±10% in the presence of EILDVPST but not in the presence of control peptide EIDVLPST. When VLA-4 or VCAM-1 was blocked, more mononuclear cells rolled on early lesions at significantly higher (approximately doubled) rolling velocities. These data demonstrate that (1) blockade of VCAM-1 can abrogate the majority (75±12%) of VLA-4–dependent monocyte adhesion on early atherosclerotic endothelia and (2) ILDV peptide interferes with VLA-4 binding to both VCAM-1 and FN and may be useful in limiting monocyte adhesion to atherosclerotic lesions. (Circ Res. 2000;87:153-159.)