The Dipteran neuroendocrine complex, consisting of the brain and ring gland, synthesizes hormones that control molting, metamorphosis, vitellogenesis and reproduction. Radiochemical assays demonstrated that juvenile hormone bisepoxide (JHB3) was the major biosynthetic product of ring glands in the higher Dipteran larvae, Drosophila melanogaster, and Sarcophaga bullata. Biosynthetic rates were shown to vary during the penultimate larval stadium, indicating a possible role for JHB3 in metamorphosis and molting. Brains, when attached to ring glands, inhibit JHB3 biosynthesis, indicating a possible neural allatostatic factor. D. melanogaster PISCF-allatostatin, and Manduca sexta allatotropin have been shown to inhibit and stimulate rates of juvenile hormone biosynthesis, but were incapable of modulating JHB3 biosynthesis in these Dipteran species. Farnesoic acid, precursor to juvenile hormone III (JH III) and methyl farnesoate (MF), inhibited JHB3 biosynthesis, but stimulated JH III and MF biosynthesis. This implies that JHB3 may utilize a different biosynthetic pathway than is currently believed.