An acridine orange staining technique was evaluated in comparison with other well-known methods for the laboratory diagnosis of leishmaniasis. A higher number of promastigotes was found in Novy-MacNeal-Nicolle (NNN) cultures inoculated with canine bone marrow, when culture samples were stained with acridine orange vital stain, compared with those detected using either Giemsa staining or unstained wet mount examination. Based on our data the acridine orange stain is a useful and timely technique in reflecting the true numbers of microorganisms present in a culture and also enhances the visualization of the parasites. The present results warrant further studies with human samples from suspected leishmaniasis patients.