One hundred years ago, Giemsa's stain was employed for the first time for malaria diagnosis. Giemsa staining continues to be the method of choice in most malarious countries, although, in the recent past, several alternatives have been developed that exhibit some advantages. Considerable progress has been made with fluorescent dyes, particularly with Acridine Orange (AO). The literature on the discovery, development and validation of the AO method for malaria diagnosis is reviewed here. Compared with conventional Giemsa staining, AO shows a good diagnostic performance, with sensitivities of 81.3%-100% and specificities of 86.4%-100%. However, sensitivities decrease with lower parasite densities, and species differentiation may occasionally be difficult. The most notable advantage of the AO method over Giemsa staining is its promptness; results are readily available within 3-10 min, whereas Giemsa staining may take 45 min or even longer. This is an important advantage for the organization of health services and the provision of effective treatment of malaria cases. The national malaria control programme of Tanzania, together with the Japan International Co-operation Agency, began to introduce the AO method in Tanzania in 1994. So far, AO staining has been introduced in 70 regional and district hospitals, and 400 laboratory technicians have been trained to use the method. The results of this introduction, which are reviewed here and have several important implications, indicate that AO is a viable alternative technique for the laboratory diagnosis of malaria in highly endemic countries.