Primary cultures of human astrocytes, expressing glial fibrillary acidic protein (GFAP), were obtained from postmortem brain tissue samples. These cultured astrocytes produced an extracellular matrix (ECM), containing laminin (Ln) and fibronectin (Fn), as shown with specific antibodies. The perinuclear staining observed in these cells indicated that these proteins were de novo synthesized. Monoclonal antibody (mAb) 90.45, which recognizes the CS1 sequence found in an alternatively spliced form of Fn, also stained cultured astrocytes. Immunohistochemical analysis of normal human brain tissue showed positive staining for the CS1 domain, both on protoplasmic and fibrous astrocytes located in the gray and white matter. In contrast to cultured astrocytes, no immunoreactivity for Ln or Fn was found on astrocytes in normal human brain tissue. These in situ data indicate that the CS1 domain expressed by astrocytes is not part of a splicing variant of Fn. Western blot analysis confirmed that the CS1 domain expressed by cultured human astrocytes is part of an astrocyte protein which is different from human Fn. The CS1 domain is a known ligand for the adhesion receptor alpha4beta1 (VLA-4). We found that the human lymphoma cell lines Jurkat and Ramos, which express alpha4beta1, bound to cultured human astrocytes, and that this interaction could be partly blocked by mAb 90.45 or a synthetic CS1 peptide. Thus, the novel CS1-containing surface protein expressed by astrocytes in vitro and in vivo, contributes to binding of lymphoblasts, and therefore may be a relevant adhesion molecule for the recruitment of alpha4-integrin expressing leukocytes into the central nervous system (CNS).