An indirect immunofluorescence (IIF) test was performed with human sera to detect cross-reactivity of Babesia microti antibodies with other species of Babesia parasites, with other blood and tissue parasites, and with various tick-borne organisms. Antisera to B. microti cross reacted with other Babesia species, but at lower dilutions than with the homologous antigens, and occurred most often during the acute phase of the disease. Cross-reactions with antibodies to malaria, Colorado tick fever, and a variety of other parasitic diseases were uncommon. However, acute and convalescent phase sera from 4 patients with suspected or confirmed Rocky Mountain spotted fever showed a rise in titer to B. microti antigen. In addition, 6 of 185 serum samples from children on an Indian reservation in North Carolina had IIF titers of ≥1:256, suggesting a possible focus of B. microti infections in humans.