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The value and role of the acridine orange/microhematocrit tube method (quantitative buffy coat [QBC®] analysis) in the diagnosis of malaria remains controversial. To establish the true sensitivity of this test in comparison with the thick blood film, we studied 49 subjects who were experimentally infected with Plasmodium falciparum in 10 malaria vaccine and infectivity trials. Diagnosis was made by the acridine orange staining method 1–3 days earlier than by the thick blood film in 23 subjects (47%) and at the same time as the thick blood film in 20. On the other hand, diagnosis was made by thick blood film earlier than by the acridine orange staining method in six individuals. There were no false positive results using acridine orange among 584 specimens studied. Diagnosis was made using acridine orange at a parasitemia of less than 11 parasites/µl of blood in 65% of cases. Where available, the acridine orange assay is clearly preferable in terms of speed and accuracy to the thick blood film for diagnosis with parasitemias of less than 150/µl of blood, and perhaps as important, for ruling out infection with P. falciparum in a symptomatic patient.