The most prominent cell-surface integrin alpha4beta1 partner, a 70-kDa protein, was isolated from MOLT-4 T leukemia cells, using anti-alpha4beta1 integrin antibody-coated beads. By mass spectrometry, this protein was identified as EWI-2, a previously described cell-surface partner for tetraspanin proteins CD9 and CD81. Wild-type EWI-2 overexpression had no effect on MOLT-4 cell tethering and adhesion strengthening on the alpha4beta1 ligand, vascular cell adhesion molecule-1 (VCAM-1), in shear flow assays. However, EWI-2 markedly impaired spreading and ruffling on VCAM-1. In contrast, a mutant EWI-2 molecule, with a different cytoplasmic tail, neither impaired cell spreading nor associated with alpha4beta1 and CD81. The endogenous wild-type EWI-2-CD81-alpha4beta1 complex was fully soluble, and highly specific as seen by the absence of other MOLT-4 cell-surface proteins. Also, it was relatively small in size (0.5 x 10(6) Da to 4 x 10(6) Da), as estimated by size exclusion chromatography. Overexpression of EWI-2 in MOLT-4 cells caused reorganization of cell-surface CD81, increased the extent of CD81-CD81, CD81-alpha4beta1, and alpha4beta1-alpha4beta1 associations, and increased the apparent size of CD81-alpha4beta1 complexes. We suggest that EWI-2-dependent reorganization of alpha4beta1-CD81 complexes on the cell surface is responsible for EWI-2 effects on integrin-dependent morphology and motility functions.