The attachment of Toxoplasma gondii to target cells is mediated by recognition of cellular heparan sulfate proteoglycans (HSPGs). The present study was performed to determine whether SAG1 and SAG3, two of the parasite surface antigens anchored to the membrane via glycosylphosphatidylinositol groups (GPIs), are involved in the tachyzoite binding to proteoglycans. The use of recombinant soluble forms of these proteins allowed us to demonstrate that SAG3, but not SAG1, interacts specifically with cellular HSPGs. Indeed, soluble recombinant SAG3 protein (recSAG3) was found to bind to immobilized heparin, whereas recSAG1 did not interact with this glycoaminoglycan. The specific adherence of recSAG3 to CHO cells was inhibited by soluble glycoconjugates, of which heparin, fucoidan and dextran sulfate were the most effective. Moreover, binding of recSAG3 to two HSPGs-deficient cell mutants was reduced by up to 80%. Proteoglycan sulfation was critical for SAG3 adherence to HSPGs as incubation of cells in the presence of sodium chlorate drastically reduced the recSAG3 binding. Finally, preincubation of CHO cells with recSAG3 blocked the adsorption of radiolabelled Toxoplasma tachyzoites. Taken together, these results indicate that SAG3 is a first glycoaminoglycan-binding protein associated with Toxoplasma, and SAG3-HSPGs interactions are involved in the parasite attachment to target cells.