Four aminopeptidase N (APN) isoforms, TnAPN1, TnAPN2, TnAPN3 and TnAPN4, were identified from the cabbage looper, Trichoplusia ni, by cDNA cloning. The deduced amino acid sequences of the four APNs indicate that TnAPN1, TnAPN2, TnAPN3 and TnAPN4 are synthesized as pre-proteins of 110, 106, 114 and 108 kDa, respectively. Sequence features of the T. ni APNs include the presence of a signal peptide at their N-termini and a prepeptide at the C-termini for the GPI anchor, the zinc binding/gluzincin motif HEX2HX18E, the gluzincin aminopeptidase motif GAMENWG and the presence of glycosylation sites. After removal of the signal peptide and the C-terminal prepeptide, the predicted molecular weights of TnAPN1, TnAPN2, TnAPN3 and TnAPN4 are 106, 102, 110 and 104 kDa, respectively. Enzymatic activity assays of various larval tissues showed that aminopeptidase activities were mainly localized in the midgut and the specific enzyme activity per mg of midgut tissue proteins was constant in T. ni larvae regardless of the composition of dietary proteins and amino acids. Both enzyme activity assays and RT-PCR analyses for the expression of the APN genes in T. ni larval tissues demonstrated that APN genes were expressed in Malphigian tubules in addition to the midgut, which was the first observation that APNs were also synthesized in insect Malphigian tubules. The finding of APN gene expression and enzyme activity in the Malphigian tubules indicated the biochemical and functional similarity of the insect Malphigian tubules to the mammalian counterpart, the kidney, in which APNs are known to play important functions.