Monkey blood samples were collected from 214 monkeys relocated as part of the wildlife rescue organized in French Guiana during the filling of the Petit Saut Dam on the Sinnamary River. These samples were tested for malaria parasites by microscopy of thick blood films and by nested PCR for small subunit rRNA genes (SSUrRNA). Parasitic blood forms similar to Plasmodium brasilianum were detected in 4 monkey species: Alouatta seniculus macconnelli, Saguinus midas midas, Pithecia pithecia and Ateles paniscus paniscus, with the highest prevalence in Alouatta monkeys. PCR was more sensitive than the conventional method for detecting low-grade parasitaemia in positive monkeys. The examination of blood films indicated that 5·6% of the animals carried parasites whereas the nested PCR for ribosomal DNA indicated a prevalence of 11·3%. The P. brasilianum SSUrRNA gene sequence was analysed and aligned with those from P. malariae, P. falciparum and P. vivax. This suggested that P. brasilianum and P. malariae are very closely related. Similar results were obtained from analysis of the sequences in P. malariae and P. brasilianum isolates of a polymorphic gene fragment analogous to the merozoite surface protein-1 (MSP-1) gene of P. falciparum. The P. brasilianum/P. malariae sequences were more similar to those of P. vivax than to those of P. falciparum, at least in the gene region examined. The high degree of DNA homology in the sequences of the SSUrRNA and msp1-like genes is consistent with other characterizations demonstrating a taxonomic relationship between P. brasilianum and P. malariae species. Our results provide further evidence that P. brasilianum and P. malariae are virtually identical and should probably be considered to be a single malaria species. This raises the question as to whether monkeys living in the rainforest are natural reservoirs for both simian and human malaria. These results have implications for the interpretation of the current epidemiological situation in French Guiana.