An indirect immunofluorescent antibody test was used to detect antibody to Babesia microti in human sera. Nine patients from Nantucket Island, Massachusetts infected with B. microti had serum titers ≥1,024. Of 84 control sera from New York City residents, 246 sera from patients with possible exposure to ticks, and 36 sera from patients with suspected or confirmed tick bites, none was reactive at titers of 1:16 or above. The within-test reproducibility was within one fourfold dilution in 95% of trials. Test-to-test reproducibility was within one fourfold dilution in 33% of trials and within two fourfold dilutions in 100% of trials. Although cross-reactions among infected patients' sera and antigens of B. argentina, B. equi, B. bigemina, Plasmodium vivax, P. falciparum, and P. brasilianum were common, titers were highest to the homologous antigen.