We report the detection of Theileria annulata, the causative agent of tropical theileriosis, by PCR in blood samples obtained from carrier cattle. The assay employs primers specific for the gene encoding the 30-kDa major merozoite surface antigen of T. annulata. A 721-bp fragment was amplified from blood samples taken monthly from calves experimentally infected with one of four different stocks of T. annulata originating in either Mauritania, Portugal, Spain, or Turkey. At the end of the experiment, five animals carried the infection for 12 months and two animals remained infected for 15 months. DNAs from six other Theileria species, T. parva, T. mutans, T. sergenti, T. buffeli, T. velifera, and T. taurotragi, were not amplified. Moreover, DNAs from four other hemoparasites (Anaplasma centrale, Anaplasma marginale, Babesia bovis, and Babesia bigemina) were also not amplified. As a control, primers derived from the small subunit rRNA gene of Theileria spp. amplified a 1.1-kb DNA fragment from all Theileria species examined but not from the other four hemoparasites. As few as two to three parasites per microliter of infected blood in a 50-microliters sample volume were detected by Southern or microplate hybridization with a T. annulata-specific cDNA probe. In addition, 92 field samples obtained from cattle in Spain were tested; 22% were positive in blood smears, 40% were positive by immunofluorescent antibody test, and 75% were positive for T. annulata by PCR. The method provides a useful diagnostic tool for detecting T. annulata carrier cattle.