This report describes the characterization and phylogenetic analysis of the deduced amino acid sequences of methionine aminopeptidase 2 (MetAP-2) enzymes from microsporidian species and genotypes of the genus Encephalitozoon. Fragments of DNA encoding 318 to 335 amino acid residues of the MetAP-2 genes were isolated from genomic DNA prepared from cultured spores of Encephalitozoon hellem, Encephalitozoon intestinalis, and Encephalitozoon cuniculi genotypes I-III. Sequence comparisons of the deduced amino acid residues indicated that the microsporidian sequences are MetAP-2-like rather than MetAP-1-like. Alignments demonstrated that the new Encephalitozoon sequences included sequences and structures conserved in eukaryotic MetAP-2s, including the five conserved, active site residues, Asp, Asp, His, Glu, and His, considered to be critical for catalysis and for coordinating the cation (e.g., cobalt) co-factor, and included residues known to interact with the antibiotic, fumagillin. The primary structure of the Encephalitozoon MetAP-2s, however, showed some dissimilarity with human and yeast MetAP-2s, including the absence of the NH2-terminal polylysine tract. Phylogenetic comparison of these Encephalitozoon MetAP-2s with orthologues from related species and from other informative taxa confirmed that the MetAP-2s of these Encephalitozoon species and strains are closely related to each other and cluster with MetAP-2s.