The in vitro secretion of ecdysteroids from the prothoracic glands of last instar larvae of Spodoptera littorulis was detected and analysed by HPLC-RIA. The primary product was identified as 3-dehydroecdysone (= 82%), with lesser amounts of ecdysone (= 18%). Interconversion of ecdysone and 3-dehydroecdysone by prothoracic glands was not detectable.3-Dehydroecdysone 3P-reductase activity was demonstrated in the haemolymph. Ecdysone, the endproduct, was characterised by reverse-phase and adsorption HPLC, chemical transformation into ecdysone 2, 3-acetonide, and mass spectrometry. The conditions for optimal activity were determined. The enzyme requires NADPH or NADH as cofactor and K,,, values for NADPH and NADH were determined to be 0.94 pM and 22.8 pM, respectively. Investigation of the kinetic properties of the enzyme, using either NADPH or NADH as cofactor, revealed that it exhibits maximal activity at low 3-dehydroecdysone substrate concentrations, with a drastic inhibition of activity at higher concentrations (> 5 pM). The results suggest that the 3-dehydroecdysone 3P-reductase has a high-affinity (low K,) binding site for 3-dehydroecdysone substrate, together with a lower-affinity inhibition site.The 3P-reductase enzyme was purified to homogeneity using a combination of poly(ethy1ene glycol) 6000 precipitation and successive FPLC fractionation on Mono-Q, phenyl Superose (twice), and hydroxyapatite columns. The native enzyme was shown to be a monomer with molecular mass of 36 kDa by SDSPAGE and gel-filtration chromatography. Furthermore, the activity of the enzyme during the last larval instar was found to reach a peak prior to that of the haemolymph ecdysteroid titre, supporting a role for the enzyme in development.Keywords: 3-dehydroecdysone 3P-reductase; ecdysone ; prothoracic gland; Spodoptera littoralis.In immature stages of insects, the prothoracic glands are the primary source of ecdysteroid (moulting hormone), generally ecdysone (Scheme 1, I) in most species. However, recently it has been shown that in most lepidopteran species studied, the major product of the glands is 3-dehydroecdysone (Scheme 1, U), together with varying proportions of ecdysone [l, 21. After release from the prothoracic glands, the 3-dehydroecdysone is reduced to ecdysone by an NAD(P)H-linked 3-dehydroecdysteroid 3p-reductase in the haemolymph [2-41. The level of reductase activity in different insect species would appear to be related to the proportion of 3-dehydroecdysone secreted by the prothoracicThe exact details of ecdysteroid biosynthesis are not fully elucidated, although the early and late stages are the best understood [5-81. There is substantial evidence for the intermediacy of 2,22,25-trideoxyecdysone (5p-ketodiol ; Scheme 1, 111) in the biosynthesis of ecdysone [5,8, 91. This labelled substrate is effi-