Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.