Duffy binding protein (DBP), a leading malaria vaccine candidate, plays a critical role in Plasmodium vivax erythrocyte invasion. Sixty-eight of 366 (18.6%) subjects had IgG anti-DBP antibodies by enzyme-linked immunosor bent assay (ELISA) in a community-based cross-sectional survey in the Brazilian Amazon Basin. Despite continuous exposure to low-level malaria transmission, the overall seroprevalence decreased to 9.0% when the population was reexamined 12 months later. Antibodies from 16 of 50 (36.0%) subjects who were ELISA-positive at the baseline were able to inhibit erythrocyte binding to at least one of two DBP variants tested. Most (13 of 16) of these subjects still had inhibitory anti-bodies when reevaluated 12 months later. Cumulative exposure to malaria was the strongest predictor of DBP seroposi-tivity identified by multiple logistic regression models in this population. The poor antibody recognition of DBP elicited by natural exposure to P. vivaxin Amazonian populations represents a challenge to be addressed by vaccine development strategies.