Chondroitin sulfate (CS) A is a key receptor for adhesion of Plasmodium falciparum-infected erythrocytes (IEs) in the placenta and can also mediate adhesion to microvascular endothelial cells. IEs that adhere to CSA express var2csa-type genes, which encode specific variants of the IE surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1). We report direct binding of native PfEMP1, isolated from IEs and encoded by var2csa, to immobilized CSA. Binding of PfEMP1 was dependent on 4-O-sulfated disaccharides and glucuronic acid rather than iduronic acid, consistent with the specificity of intact IEs. Using immobilized CS oligosaccharides as neoglycolipid probes, the minimum chain length for direct binding of PfEMP1 was eight monosaccharide units. Similarly for IE adhesion to placental tissue there was a requirement for 4-O-sulfated GalNAc and glucuronic acid mixed with non-sulfated disaccharides; 6-O-sulfation interfered with the interaction between placental CSA and IEs. The minimum chain length for maximal inhibition of adhesion was 10 monosaccharide residues. Partially 4-O-sulfated CS oligosaccharides (45-55% sulfation) were highly effective inhibitors of placental adhesion (IC(50), 0.15 microg/ml) and may have potential for therapeutic development. We used defined P. falciparum isolates expressing different variants of var2csa in adhesion assays and found that there were isolate-specific differences in the preferred structural motifs for adhesion to CSA that correlated with polymorphisms in PfEMP1 encoded by var2csa-type genes. This may influence sites of IE sequestration or parasite virulence. These findings have significant implications for understanding the pathogenesis and biology of malaria, particularly during pregnancy, and the development of targeted interventions.