Four distinct epitopes (A, B1, B2, and C) have been functionally defined on the human alpha4 integrin. In this study, two cross-reactive antihuman alpha4 monoclonal antibodies (mAb) (HP2/1 and HP2/4 specific for epitopes B1 and B2, respectively) were used to functionally characterize the rat VLA-4 subunit and to define similar functional epitopes in this rodent species. It was found that B1 and B2 anti-alpha4 mAb completely block adhesion to fibronectin, but the inhibition of adhesion to vascular cell adhesion molecule-1 (VCAM-1) with HP2/1 mAb was lower than with HP2/4 mAb. It was also observed that epitope B2 HP2/4 mAb induced homotypic aggregation in rat lymphocytes, whereas epitope B1 HP2/1 mAb did not. Using the HgCl2 model of nephritis, this study shows the protective effect of both anti-alpha4 mAb against infiltration of the renal interstitium by leukocytes. Nevertheless, HP2/1 mAb, but not HP2/4 mAb, virtually abolished the anti-glomerular basement membrane antibody synthesis and glomerular deposits. These findings indicate the dual but independent role played by alpha4 integrins in both extravasation of leukocytes and in the production of antibodies. Finally, this study demonstrates that anti-rat VCAM-1 mAb showed a positive reactivity of the renal vascular endothelium and, most importantly, that administration of anti-VCAM-1 antibodies completely abrogated the interstitial cell infiltrates without affecting anti-glomerular basement membrane antibody production. These results confirm the important role played by VLA-4/VCAM-1 pathway in leukocyte infiltration, and further support the dual and independent role of alpha4 integrins in both renal infiltration and autoantibody synthesis in this model of renal disease.