Cross-species transmission of simian foamy virus (SFV) to human beings from chimpanzees, baboons, and African green monkeys has been described. Although macaques are the non-human primate most often handled in research, human infection with SFV from macaques has not been reported. Two of 46 primate-facility workers tested positive for antibodies that reacted with an immunoblot that contained macaque foamy virus antigens. Phylogenetic assessment of a 96-bp fragment of amplified proviral DNA isolated from peripheral-blood mononuclear cells from one infected individual was consistent with SFV infection of macaque origin. Frequent use of macaques in biomedical research, and identification of persistent retroviral infection from macaques to human beings, could have implications for public-health policy and occupational health and safety.