Region II of the malaria circumsporozoite (CS) protein is highly conserved between the CS proteins of different species of malaria. Amino acid sequences homologous to that of region II are found in thrombospondin, properdin, von Willebrand factor and a few other proteins. We show here that the native CS protein from the rodent parasite Plasmodium berghei, and recombinant Plasmodium vivax and Plasmodium falciparum CS proteins containing region II, but not recombinant proteins lacking region II, specifically bind to sulfatides and cholesterol-3-sulfate. The binding is abolished following reduction and alkylation of the proteins. Region II contains 2 cysteines separated by only 3 amino acids, S(N), V, T, and these are the only cysteines present in our recombinant proteins. Therefore, our findings strongly suggest that the region II cysteines are linked by a disulfide bond forming a small peptide loop. We also present evidence that the recognition of sulfatides, cholesterol-3-sulfate, or other cross-reactive sulfated macromolecules by region II may be required during sporozoite invasion of liver cells. Antibodies to a peptide representing region II react with live sporozoites and with sporozoites fixed with glutaraldehyde, indicating that this region is exposed on the surface of the parasites. Furthermore, we have found that the sulfatide and cholesterol-3-sulfate recognition by the CS proteins, and the invasion of hepatocytes by P. berghei sporozoites, are specifically inhibited by dextran sulfate.