ABSTRACT.Incubation conditions have been established for the corpus cardiacum‐corpus allatum (CC‐CA) complex of femalePhormia regina(Meigen), which will support CC‐CA biosynthetic activitiesin vitroas measured by the incorporation of a labelled methyl group with L‐[methyl‐3H]methionine as the methyl donor. After incubation, radioactivity in the organic extract of the medium was determined by scintillation counting. Analysis of the organic extract with reverse phase high‐performance liquid chromatography (HPLC) revealed that a compound which has similar retention time (UV absorbance) with synthetic JH III was synthesized by the CC‐CA complexes of liver‐fed females. By using this short‐term,in vitro, radiochemical assay for CA activity, it was shown that a protein diet significantly increases the activity of the CA compared with females fed only a sugar‐water diet. Furthermore, use of HPLC separation, in conjunction with scintillation counting of time‐collected fractions, demonstrated the existence of a moiety containing incorporated radiolabeled methyl group (from the methionine) which did not co‐elute with JH I or JH III standards. These results suggest that inP. reginause of the incorporation of a radiolabeled methyl group to measure JH biosynthesis (CA activity) can be misleading if the compounds which do not co‐elute wi