The diagnosis of louse-borne relapsing fever is commonly made on the basis of the detection of Borrelia spirochetes on Giemsa-stained thin blood films. In the present study, we used acridine orange-coated quantitative buffy coat (QBC) tubes, centrifugation, and fluorescence microscopy to detect Borrelia. Between July and August 1998, we used the QBC technique to diagnose 7 patients with borreliosis who visited a rural clinic in southwest Ethiopia. In laboratory studies that used Borrelia burgdorferi as a model, we detected spirochetes at concentrations as low as 10 organisms/mm3, whereas the number of positive readings assessed by means of stained blood films fell significantly at dilutions below 3,263 organisms/mm3. The greater sensitivity of the QBC technique is important in areas where Borrelia is endemic, as in the Horn of Africa. It may also prove useful in evaluating relapsing fevers in travelers.