Virgin females of the Japanese giant looper (Ascotis selenaria cretacea, Assc) in the family of Geometridae secrete an epoxyalkenyl sex pheromone to attract males. To regulate its biosynthesis in the pheromone gland, Assc females produce a pheromone biosynthesis-activating neuropeptide (PBAN) in the suboesophageal ganglion (SG), as do females in many lepidopteran species. We have isolated Assc-PBAN cDNA, which encodes 181 amino acids, including a PBAN homologue and four other putative peptides: a diapause hormone (DH) homologue, alpha-SG neuropeptide (SGNP), beta-SGNP, and gamma-SGNP, all of which shared an FXPR(K)L motif on their C-termini. Although PBANs with 30-35 amino acids have been characterized from 15 other species, the Assc-PBAN homologue consisted of 28 amino acids and showed low homology (<46%) compared with the others. Assc-beta-SGNP with eight amino acids was also shorter than the other beta-SGNPs (16-22 amino acids). Furthermore, all of the known PBAN cDNAs have a GRR sequence between beta-SGNP and PBAN as a cleavage site, but the Assc-PBAN cDNA showed an unusual GR sequence at the corresponding position, indicating the possibility of non-cleavage between the beta-SGNP and PBAN. When the GR sequence was a cleavage site, the question arose of whether or not the glutamine residue at the N-terminus of the Assc-PBAN homologue was cyclized. To identify the sequence of the Assc-PBAN, the brain-SG extract was fractionated by HPLC referring to three synthetic peptides with the predicted sequences. The chromatographic behavior of the natural pheromonotropic peptide revealed the unique structure of Assc-PBAN including beta-SGNP, i.e., SVDFTPRLGRQLVDDVPQRQQIEEDRLGSRTRFFSPRL-NH(2), as the first determination of PBAN from the insects producing an epoxyalkenyl sex pheromone.