The specific diagnosis of babesiosis, which is caused by the piroplasm Babesia microti, is made by microscopic identification of the organism in Giemsa-stained thin blood smears, detection of babesial antibody in acute-and convalescent-phase sera, or identification of the organism following the injection of patient blood into laboratory animals. Although rapid diagnosis can be made with thin blood smears, parasites are often not visualized early in the course of infection. PCR is a new, rapid diagnostic technique for the detection of Babesia spp. that has not yet been systematically evaluated. We conducted a blinded study of the sensitivity, specificity, and reproducibility of the PCR-based test with patients with babesiosis and a group of asymptomatic subjects residing in a region in southern New England where babesiosis is enzootic. Among 19 patients with recent babesial illness, we found that PCR was as sensitive and specific as the use of Giemsa-stained blood smears and inoculation of hamsters. Among asymptomatic subjects, the PCR result was positive for 3 persons with recent babesial infection and was negative for 41 persons without previous babesial infection. We conclude that the B. microti PCR procedure is sufficiently sensitive, specific, and reproducible for use in the diagnosis of acute babesiosis.