This paper reports the first isolation of Mayaro (MAY) virus from a patient infected in French Guiana. The identification was initially performed using immunofluorescent antibody testing with specific mouse antibody, and confirmed by plaque-reduction neutralization testing and reverse transcription-polymerase chain reaction. To determine if MAY virus infection is widespread in French Guiana, a serosurvey was performed to determine the prevalence of antibody to this virus in various ethnic groups and areas of French Guiana. Human sera (n = 1,962) were screened using the hemagglutination inhibition (HI) test. To determine whether MAY virus circulates in the rain forest, a serosurvey in monkey populations was performed. Monkey sera (n = 150) were also screened for antibody to MAY virus using HI testing. Of the human sera tested, 6.3% were positive for anti-MAY virus antibodies. Significant differences in MAY virus seroprevalence between different age groups were observed. Seroprevalence rates increased with age, with a large increase in people 10-19 years of age in comparison with those less than 10 years of age. After adjustment for age, significant differences were also found between places of residence. The prevalence of anti-MAY virus antibody was higher in people living in contact with the forest, especially in the Haut Oyapock area (odds ratio [OR] = 97.7, 95% confidence interval [CI] = 48.2-197.9) and along the Maroni River (OR = 39.7, 95% CI = 20.6-76.6). The ethnic differences observed in this study were probably due to differences in residence. Among monkeys, higher seroprevalence rates were found in Alouatta seniculus (66.0%) than in Saguinus midas (18.2%). Among Alouatta, the seroprevalence increased significantly with weight (and therefore with age). This study indicates that MAY virus is present in French Guiana, and human infections occur in areas where people live near the tropical rain forest.