Midgut aminopeptidases N (APNs) play important physiological roles in protein digestion and serve as receptors for certain Bacillus thuringiensis toxins. By taking approaches of both midgut expressed sequence tags sequencing and RT-PCR based cDNA cloning, we have identified and cloned full length cDNAs for four APNs from cabbage looper, Trichoplusia ni. The cDNAs encode APNs with predicted molecular weights of about 110 kDa with characteristic midgut aminopeptidase N features. The gene expression was primarily localized to the midgut cell membranes by both biochemical analysis of enzymatic activities and RT-PCR analysis of the APN gene transcripts.