Two juvenile hormones are isolated from organ cultures of corpora allata of the tobacco hornworm moth, Manduca sexta Johannson, and are purified by high-resolution liquid chromatography. These are identified as methyl (2E, 6E)-(IOR)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate, a new natural hormone, and methyl (2E,6E) -(IOR,11S) -10,11 -epoxy -3,7,11 -trimethyl- Continued improvements in insect tissue culture techniques have led to their increased use in studies of endocrine physiology (5). Indirect indications have been obtained that corpora allata can survive and function in vitro (6), and recently cecropia juvenile hormone has been recovered and identified from cultures of brain-corpora cardiaca-corpora allata complexes of H. cecropia (7).We report both an in vitro system that cleanly produces two hormones 2 and 3 from corpora allata of Manduca sexta, and the greatly simplified purification and chemical identification of the active hormones, one of them identified for the first time as a natural product of insects. Preparation of Cultures. Adult female moths, 0-48 hr after eclosion, were anesthetized with CO2 and surface sterilized by immersion in 70% ethanol; the cuticular scales and hairs over the head and thorax were rubbed off with paper towel. The dorsal body wall over the head and cervical membrane was cut away, taking care not to bring the external surface into contact with the underlying tissues. The corpus cardiacum-corpus allatum complex was located attached to either side of the aorta posterior to the brain, and was removed by cutting the aorta, nerves, and tracheae in front of and behind the glands. Groups of excised glands were transferred through four successive baths of culture medium, the first two containing 1 mM recrystallized phenylthiourea, and then into a sterile Leighton culture tube containing 0.5 ml of fresh culture medium.During incubation at 20-25', the culture medium was replaced at 2-to 8-day intervals with a total of three changes over a 15-day period. The medium removed from the cultures was either extracted immediately or stored for up to 2 weeks at -20°.Culture Medium. We used Grace's Insect T.C. Medium (GIBCO) without methionine, supplemented with 1% (w/v) bovine plasma albumin (fraction V, Metrix) and L-[methyl-14C]methionine (ICN, 48.4 Ci/mol) to a final concentration of 0.04 mM, containing 2,uCi/ml. Extraction Procedure. Culture media were extracted three times by vigorous shaking with equal volumes of highly purified ethyl acetate. The pooled organic phases were reduced to a small volume under N2, applied to washed (hexaneethyl acetate 1:1) thin-layer chromatography plates (silica gel GF-254, Brinkmann, 0.25 mm thickness), and developed in hexane-ethyl acetate 1:1 (v/v). Chromatoplates were radioscanned (Packard model 7201) and appropriate zones were eluted with pure ethyl acetate and stored in that solvent at -200.Chromatography and Spectroscopy. High-resolution liquid chromatography separations were performed on a homemade instrument incorporating a Haskel E...