We raised specific antisera against recombinant VAR2CSA domains produced in Escherichia coli and in insect cells. All were reactive in enzyme-linked immunosorbent assay, but only insect cell-derived constructs induced immunoglobulin G (IgG) that was reactive with native VAR2CSA on the surface of infected erythrocytes. Our data show that five of the six VAR2CSA Duffy-binding-like domains are surface exposed and that induction of surface-reactive VAR2CSA-specific IgG depends critically upon antigen conformation. These findings have implications for the development of vaccines against pregnancy-associated Plasmodium falciparum malaria.