The ability of viruses and bacteria to interact with the extracellular matrix plays an important role in their infectivity and pathogenicity. Fibronectin is a major component of the extracellular matrix in lymph node tissue, the main site of HIV deposition and replication during the chronic phase of infection. Therefore, we asked whether matrix fibronectin (FN) could affect the ability of HIV to infect lymphocytes. To study the role of matrix FN on HIV infection, we used superfibronectin (sFN), a multimeric form of FN that closely resembles in vivo matrix FN. In this study we show that HIV-1IIIB efficiently binds to multimeric fibronectin (sFN) and that HIV infection of primary CD4+ lymphocytes is enhanced by >1 order of magnitude in the presence of sFN. This increase appears to be due to increased adhesion of viral particles to the cell surface in the presence of sFN, followed by internalization of virus. Enzymatic removal of cell surface proteoglycans inhibited the adhesion of HIV-1IIIB/sFN complexes to lymphocytes. In contrast, Abs to integrins had no effect on binding of HIV-1IIIB/sFN complexes to lymphocytes. The III1-C peptide alone also bound HIV-1IIIB efficiently and enhanced HIV infection, although not as effectively as sFN. HIV-1IIIB gp120 envelope protein binds to the III1-C region of sFN and may be important in the interaction of virus with matrix FN. We conclude that HIV-1IIIB specifically interacts with the III1-C region within matrix FN, and that this interaction may play a role in facilitating HIV infection in vivo, particularly in lymph node tissue.