The integrin very late antigen, (VLA-4) alpha 4 beta 1 and its counter receptor vascular cell adhesion molecule 1 (VCAM-1) are involved in B cell maturation and pre-B cell attachment to bone marrow stroma cells. We have analyzed whether heat-stable antigen (HSA), a marker for immature leukocytes, is involved in such cell adhesion phenomena. HSA is a glycolipid-anchored, highly glycosylated surface protein differentially expressed on cells during the maturation of both the hematopoietic and nervous systems. We found that pre-B cells lacking HSA (due to targeted disruption of both alleles) can still bind via VLA-4 to tumor necrosis factor alpha-stimulated endothelioma cells. This binding, however, cannot be blocked by an anti-VCAM-1 antibody. Restoration of HSA expression restores the inhibitable VCAM-1 binding. We also found that pre-B cells lacking HSA did not bind to the FN40 fragment of fibronectin but reexpression of HSA restored VLA-4-mediated binding to fibronectin. Thus, expression of HSA on pre-B cells modifies the binding specificity of VLA-4 for two known ligands.