Abstract The titer of juvenile hormone III (JH-III) has been determined by the use of combined gas chromatography-mass spectroscopy (GC-MS) in whole body extracts of the larvae, prepupae, pupae and adults of Drosophila hydei. A characteristic JH-III titer curve was established by use of the hormone derivatives. No other juvenile hormone homologs were detected. JH-III shows a broad peak maximum of about 30 pmol/g fresh weight during the last larval instar, whereas only traces of the hormone are detectable at pupariation. Prepupae and pupae exhibit about the same JH-III level. In older pupae and in the pharate adults there was no JH-III detectable but it reappeared soon after emergence. Low values of JH-III are found in young male and female flies. The JH contents rise to distinct peaks in older and reproductive adults, both in male and female animals.