Abstract An α-ecdysone-binding protein fraction, approx. mol. wt. 120,000, has been demonstrated in haemolymph of Drosophila hydei late third instar larvae. The protein has been partly characterized by Sephadex G-25 filtration, hydroxylapatite chromatography, density gradient centrifugation, and ezyme digestion experiments. The protein-steroid complex appears to be heat stable. Binding of labelled ecdysone to the protein fraction is significantly reduced in competition experiments using unlabelled ecdysones. An ecdysone-binding protein fraction has been detected in hand-isolated total alimentary tract tissues (predominantly midgut, Malpighian tubules, and salivary glands) and in mass-isolated midgut and Malpighian tubules. The sedimentation properties of this protein-hormone complex are similar to those of the complex found in haemolymph.