This paper describes a sero-epidemiological study of malaria prevalence in French Guiana. An immunofluorescence assay and an enzyme-linked immunosorbent assay were used to detect antibodies against blood-stage antigens and synthetic peptides mimicking the repetitive epitope of the sporozoites of Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae/brasilianum, in 218 human sera and 113 non-human primate sera collected in French Guiana. Almost all the monkey sera tested had antibodies against malaria blood-stages (98%) and a large majority (73%) also tested positive with the P. malariae/brasilianum circumsporozoite peptide. A number of primate samples also reacted positively with P. falciparum NANP repeats in a very specific manner, suggesting that monkeys in the rainforest are bitten by mosquitoes infected with human malaria parasites. Seroprevalences were lower in the humans tested but Indian tribes on the borders with Suriname and Brazil were clearly more exposed to malaria than other ethnic groups, with a prevalence of nearly 70% seropositivity. P. vivax infections accounted for much of the observed pattern of reactivity, but there was also a high frequency of positive reactions to the P. brasilianum/malariae peptide. Similarly, a large proportion of the sera obtained from Bush Negro populations tested positive for P. malariae/brasilianum repeats. These data add to the emerging evidence that non-human primates might constitute a natural reservoir, not only for simian, but also for human malaria, and therefore suggest that they might be responsible for the maintenance of foci of P. malariae, and possibly of other malaria species, in isolated areas of the Amazonian rainforest.