Using the hydroxyapatite binding assay, a juvenile hormone III (JH III) binding protein has been identified in the hemolymph of postfeeding third instar larvae of Drosophila melanogaster. This binding component was shown by photoaffinity labeling and gel filtration to be a single protein with a native molecular weight of nearly 400 kDa. It has an equilibrium dissociation constant for JH III of 1.5 nM, an affinity 10 times higher than that of similar proteins from other insects. It exhibits high specificity for the natural hormone, with the following order of binding affinities: JH III > JH II > JH I >; JH III acid > methophrene. It is separate from another component in the hemolymph that binds methoprene nonspecifically. The very high binding affinity and high specificity suggest that this JH III binding protein has a critical role in JH transport in the hemolymph.