Three cDNAs encoding aminopeptidases HpAPN1, HpAPN2 and HpAPN3, were isolated from a 5th instar larval midgut cDNA library from Helicoverpa punctigera, the Australian native budworm. The sequences recovered contain open reading frames encoding proteins of 1011, 952, and 1013 amino acids, respectively. All three proteins share the consensus zinc binding/gluzincin motif HEXXHX(18)E and the sequence GAMEN common to gluzincin aminopeptidases. Furthermore, signal peptide sequences and C-terminal hydrophobic regions preceded by three small amino acids qualifying for cleavage and GPI anchor attachment are present in all three protein sequences. Northern blotting results indicate differences in the levels of expression and developmental regulation of all three aminopeptidases. HpAPN1, HpAPN2, and HpAPN3 are more closely related to APNs from other lepidopterans than they are to each other. This report of three different aminopeptidases N in Helicoverpa punctigera adds support to a recent suggestion that at least one gene duplication has taken place in ancestral lepidopterans. The full sequences of the aminopeptidases are available at GENBANK with the following accession numbers: HpAPN1: AF217248, HpAPN2: AF217249, HpAPN3: AF217250.