Using purified recombinant fibronectins we show that WEHI 231 lymphoid cells spread only on fibronectin containing the alternatively spliced V region. Spreading is specifically blocked by peptides from the V25 segment (also called CS-1), which can be selectively spliced out independently of the rest of the V region. Using synthetic peptides we localize the binding site to a 10 amino acid segment that is highly conserved. Integrin α₄β₁ is a major integrin on the surfaces of these cells and binds specifically to the V25 segment with a primary specificity for the conserved 10 amino acid sequence. Antibodies to integrin α₄ inhibit spreading of WEHI 231 cells on V⁺ fibronectin. Therefore, integrin α₄β₁ is a fibronectin receptor specific for an alternatively spliced cell adhesion site and may play important roles in selective adhesion of various cell types to specific forms of fibronectin.