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Female sex workers (FSWs) are an important group of people vulnerable to sexually transmitted infections. Northern Brazil is a rural and socioeconomically underdeveloped region, with lack of epidemiological information on syphilis in key populations. This study investigated the prevalence and factors associated with exposure to Treponema pallidum among FSWs in three major cities in northern Brazil. This cross-sectional study was conducted with a convenience sample of 415 FSWs from the cities of Belém, Macapá, and Rio Branco. Blood samples and personal data were collected from January 2009 to August 2010. Rapid plasma reagin and immunoenzymatic assays were used to detect antibodies against T. pallidum. Logistic regression models were used to determine factors associated with exposure to T. pallidum. In total, 36.1% were exposed to T. pallidum, and 15.7% had active syphilis. Sexual risk behaviors, use of illicit drugs, low education, and reduced monthly income were associated with exposure to T. pallidum. The high rate of exposure to T. pallidum indicates the urgent need for measures to identify, treat, and prevent syphilis and an education program for the valuation, care, and social inclusion of FSWs in northern Brazil.