The renewed interest in the use of fluorochromes for malaria diagnosis prompted us to evaluate the acridine orange fluorescence technique on blood slides, and to compare it with established techniques using thick and thin blood films and the QBC malaria test, using the Giemsa-stained thick film technique as our standard method for comparison. We compared 123 positively diagnosed cases and 120 negative cases. For primary samples (day 0), the sensitivity of the thin blood film fluorescence acridine orange technique (AO) was 96.4%, and its specificity was 95.1%. In cases of imported malaria, with a prevalence rate of 16.2%, the positive predictive value was 79.2% and the negative predictive value 99.3%. Sensitivity of AO was significantly higher than that of Giemsa-stained thin blood films for parasitaemias < 5000/microL. The potential of AO for species diagnosis of Plasmodium was 85.2%, using Giemsa-stained thin films as the reference technique. Where QBC imposes a cost limitation, especially in developing countries, despite its high performance, the AO diagnostic technique is a valuable alternative, because of its simplicity, almost negligible cost, and its diagnostic reliability. The method may also have potential value in the diagnosis of other microbiological diseases.