A non-I-domain integrin, alpha4beta1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of alpha4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of alpha4 (which spans repeats 2-5 of the seven N-terminal repeats) with the corresponding regions of alpha5. Swapping residues 112-131 in repeat 2, or residues 237-247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40-52 in repeat 1, residues 151-164 in repeat 3, or residues 282-288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112-131, 151-164, and 186-191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published beta-propeller folding model of the integrin alpha4 subunit [Springer, T. A. (1997) Proc. Natl. Acad. Sci. USA 94, 65-72], in which seven four-stranded beta-sheets are arranged in a torus around a pseudosymmetric axis. The regions of alpha4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the beta-propeller model, although they are not adjacent in the primary structure.