Leishmania tropica is one of the main etiological agents of cutaneous leishmaniasis in Iran. For ultrastructural and isoenzyme study, axenic amastigotes were cultured in a brain-heart infusion medium containing 20 % fetal calf serum, pH 4.5, and incubated at 37 °C in 5 % CO(2). Different stages of L. tropica revealed the same isoenzyme profiles after comparing four enzyme systems including phosphoglucomutase, 6-phosphogluconate dehydrogenase, malate dehydrogenase, and nucleoside hydrolase II. Different isoenzyme patterns for glucose-phosphate isomerase, glucose-6-phosphate dehydrogenase, nucleoside hydrolase I, and malic enzyme enzymic systems were seen; thus, these isoenzyme systems among the eight systems studied were more efficient in characterizing L. tropica amastigotes. The structure of the axenic amastigotes was essentially similar to that of the promastigotes except for some important characteristics including the flagellum, flagellar pocket, paraxial rod, and the subpellicular microtubules.