Tick-borne borreliosis (relapsing fever) can be an important, unsuspected cause of febrile illness. The diagnosis is generally made by identifying Borrelia spirochetes in stained peripheral blood smears. Since Borrelia may be difficult to detect with Romanowsky stains, an alternative method, using acridine orange (AO), was used to screen blood smears. Duplicate blood smears of seven patients were examined with the AO technique and Romanowsky stains. In all seven cases spirochetes were easily identified with the AO-stained smears compared with only five cases with Romanowsky stains. In a double-blind laboratory experiment, six of ten duplicate smears from a single patient with mild spirochetemia were positive by AO, whereas only two of ten were positive by Romanowsky stain. We concluded that the AO stain is simple, rapid and more sensitive than Romanowsky methods for detecting cases of low-level spirochetemia.