Recent evidence indicates that higher dipterans biosynthesize predominantly the novel juvenile hormone (JH) bisepoxide, methyl (2E,6trans)-6,7,10,11-bisepoxy-3,7,11-trimethyl-2-dodecen oate (JHB3), together with a small proportion of methyl (2E,6E)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate (JHIII) and possibly its 6,7-epoxy isomer. A strategy of gas chromatography-mass spectrometry (GC-MS) involving cool on-column GC and negative-ion chemical ionization MS has enabled the selective analysis of underivatized JH-like products released in vitro by ring glands and corpora allata of Lucilia cuprina. JHB3 is released to the exclusion of mono-epoxidized and unepoxidized esters. The rate of JHB3 release in adult females increases dramatically after protein feeding from 0.2 to 5.2 pmol/h/gland. Values agree well with the rate of JH biosynthesis determined by radiochemical assay. As revealed by thin-layer chromatography, the radiolabel is predominantly incorporated into JHB3, of which only 2% is retained in the corpus allatum indicating almost immediate release from the gland. Approximately 5% of radioactive material retained in the glands co-migrates with JHIII and may be an intermediate in JHB3 synthesis. We conclude that JHB3 is the only juvenile hormone biosynthesized in vitro by L. cuprina. The data suggest that JHB3 may be the exclusive JH synthesized by higher dipterans.