Juvenile hormone III (JH III), a sesquiterpenoid involved in the regulation of insect development and reproduction, appears to be ubiquitous in insects and has also been isolated from some species of sedge. The quantification of JH III extracted from the sedge Cyperus iria by HPLC and by radioimmunoassay (RIA) has been compared. The RIA, developed originally to determine insect haemolymph titres, was optimized for the analysis of plant tissues. The antiserum 31867 displayed minimal cross‐reactivity with precursors of JH III from the insect biosynthetic pathway or with extracts from related plant species. Analysis of standards and plant extracts by HPLC and RIA confirmed that there was no significant difference between the ability of these two techniques to quantify JH III (paired t‐test). Therefore, RIA was subsequently used to determine the distribution of JH III in mature plants of C. iria. Substantial concentrations were detected in the above‐ground plant tissues (0.2–1.2 µg/g fresh weight), but the majority of JH III was found in the roots (43.5 µg/g fresh weight). Copyright © 2000 John Wiley & Sons, Ltd.