Corpora allata from adult female Manduca sexta biosynthesize the sesquiterpenoid juvenile hormone (JH) III and the unusual ethyl-branched homologue JH II in vitro. We maintained corpora allata in medium 199 using [methyl-(3)H]methionine as the source of the JH methyl ester moiety and as a mass marker. This allowed measurement of the relative contributions of (14)C-labeled precursors to the biogenesis of JH II and III carbon skeletons. We showed efficient incorporation of a propionate equivalent, from isoleucine or valine catabolism, into the ethyl-branched portion of JH II, using double-label liquid scintillation counting of isolated JHs and gas chromatography/mass spectrometry with selected ion monitoring of JH deuteromethoxyhydrin derivatives. Methionine was a poor source of propionate for JH II biosynthesis, while glucose, succinate, threonine, and beta-alanine did not contribute propionate at all. Leucine, isoleucine, and glucose incorporated into JH III and the acetate-derived portion of JH II.