Fatty acyl-ester hydrolase was not detectable in dry sunflower seeds using various p-nitrophenyl-acyl-esters, 1,2-O-didodecyl-rac-glycero-3-glutaric acid-resorufin ester or emulsified sunflower oil as substrate. After inhibition of the seeds, acyl-ester hydrolase activity slowly developed in cotyledon extracts and was maximal after 5 days. No activity was directly measurable on oil bodies. The enzyme was purified 615-fold to apparent homogeneity, as determined by performing SDS-PAGE electrophoresis, and biochemically characterized. With p-nitrophenyl-caprylate the optimum pH was around 8.0. The purification procedure involved an acetone powder from 5-day dark-germinated seedlings, chloroform-butanol extraction and three chromatography steps. We obtained 35 micrograms of pure enzyme from 250 g of fresh cotyledons with an activity yield of around 7%. It should be possible to subsequently improve this low recovery as we gave priority here, in the first instance, to purity at the expense of the yield. The enzyme consisted of one glycosylated polypeptide chain with a molecular mass of approx. 45 kDa and, as far as we could tell, it did not seem to require metal ions to be fully active, as it was not inhibited by EDTA or o-phenanthroline and not activated by various mono or bivalent metal ions. The amino acid composition showed the presence of four cysteine and four tryptophan residues. The enzyme was partially inhibited by dithiothreitol, DTNB and PCMB. The fact that high inhibition was observed in the presence of PMSF indicates that a serine residue may possibly be involved in the catalytic mechanism. The hydrophobicity index was about 53.6% which places this enzyme in the class of the soluble proteins in good agreement with the fact that it was mainly present in the soluble part of the crude extract. Partial characterization of glycan chains, using antiglycan antibodies, showed the presence of complex Asn-linked glycans. The enzyme was active on purified sunflower glycerol derivatives. It was also able to hydrolyze monooleyl and dioleyl glycerols, as well as phosphatidylcholine, but not cholesteryl esters. Using p-nitrophenyl-acyl-esters as substrate, the highest activity was observed with middle-chain derivatives (C6 and C8). Its maximum activity was about 0.015 units mg-1 with sunflower oil. It was not activated by an organic solvent such as isooctane. This enzyme probably is involved in acyl-ester hydrolysis which follows triacylglycerol mobilization by true lipases.