Expression of fibronectin (FN) isoforms containing CS1, a 25-amino acid sequence present within the alternatively spliced IIICS region of FN, has been analyzed in rheumatoid arthritis (RA) synovium. Unexpectedly, CS1-containing FN variants were exclusively found on endothelium but not extracellular matrix (ECM) of RA synovium. Lumenal expression of CS1 on RA endothelial cells, as observed by electron microscopy, correlated with inflammation in RA, since normal synovium expressed little CS1 without appreciable decrease in ECM FN. CS1 expression on human endothelial cells was further shown by FN mRNA analyses. In adhesion assays on frozen RA synovial sections, T lymphoblastoid cells expressing functionally activated alpha 4 beta 1 integrin specifically attached to the intravascular surface of RA endothelium. Binding was abrogated by both anti-alpha 4 integrin and CS1 peptides. Our observations suggest direct involvement of CS1-containing FN in recruitment of alpha 4 beta 1-expressing mononuclear leukocytes in synovitis, and provide basis for therapeutic intervention in RA.