The Duffy binding-like (DBL) domains are common adhesion modules present in Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variants, which are responsible for immune evasion and cytoadherence. Knowledge about how immune responses are acquired against polymorphic DBL domains of PfEMP1 can aid in the development of vaccines for malaria. A recombinant DBLalpha domain, encoded by R29 var1, which binds complement receptor 1 to mediate rosetting by the P. falciparum laboratory strain R29, was expressed in Escherichia coli, renatured by oxidative refolding to its native form, and purified to homogeneity. Antibody levels in 704 plasmas obtained from residents of areas of different levels of malaria endemicity in Orissa (India) and Manhiça (Mozambique) were assessed by enzyme-linked immunosorbent assay. The refolded DBLalpha domain was pure, homogeneous, and functional in that it bound human erythrocytes with specificity and was capable of inhibiting rosetting. The proportion of individuals who had measurable anti-DBLalpha immunoglobulin G responses was low in areas of low malaria endemicity in Orissa (6.7%) but high in areas of high endemicity in Orissa (87.5%) and Manhiça (74.5%). Seroprevalence and antibody levels against the recombinant protein increased with the age of inhabitants from areas with high transmission rates (P < 0.001). Half of the children in these areas had seroconverted by the age of 5 years. These findings suggest that in spite of the extreme polymorphism of PfEMP1 DBLalpha domains, the acquisition of specific antibodies is rapid and age related and reflects the reduced risk of malaria in areas with high transmission rates. Further studies are required to elucidate the role of these antibodies in protection from malaria.