Aminopeptidase activity was detected in Encephalitozoon intestinalis using a fluorometric assay. The aminopeptidase was capable of hydrolysing different amino acids bound to 7-amino-4-trifluoromethyl coumarin, with maximal activity against the amino acid, leucine. Aminopeptidase activity was localized in E. intestinalis spores and in intracellular stages. Enzymatic activity was inhibited by the traditional aminopeptidase inhibitors, bestatin and its analogue, nitrobestatin. Inhibition with the chelating agents, EDTA and 1,10-phenanthroline, suggested that the enzyme activity belongs to the metalloaminopeptidase class. Subcellular fractionation demonstrated that maximal enzyme activity was localized in the cytosolic fraction. Direct fluorogenic substrate analysis by native polyacrylamide gel electrophoresis estimated a molecular weight of 70·8 kDa. Direct fluorogenic analysis by polyacrylamide ampholyte gel electrophoresis indicated an isoelectric point of 4·8. The enzyme was both heat (>37 °C) and cold (<0 °C) labile with an optimal activity at pH 7·2. This is the first report characterizing a cytosolic aminopeptidase in microsporidia.