Megasomes are large lysosomes found in the amastigote stage of Leishmania species belonging to the mexicana complex. The biogenesis of megasomes was investigated by transmission electron microscopy during the transformation of promastigotes into the amastigote form of L. amazonensis maintained in axenic cultures. Mainly small vacuoles with low electron density were found in the promastigote and early intermediate forms. Morphometrical analysis showed an increase in the volume density of these structures during the transformation process. Cysteine proteinase was localized in this structure by immunocytochemical assay. Membrane-bounded structures filled with electron-dense material were also found in significant amounts from the 2nd day on. These structures were relatively abundant, both in axenic and lesion-derived amastigotes, but not in stable long-term axenic amastigote culture. A three-dimensional reconstruction of lesion-derived amastigotes and axenic amastigotes of L. amazonensis demonstrated that megasomes comprise almost 5% of the total cell volume. In addition, the development of other organelles was examined during the transformation process.