An olive beta-glucosidase was purified to apparent homogeneity from mature fruits ( Olea europaea cv. Picual) by selective extraction and successive anion exchange and hydrophobic interaction chromatographic procedures. The enzyme was shown to be a homodimer made up of two identical subunits of 65.4 kDa. Optimum activity was recorded at pH 5.5 and 45 degrees C. The enzyme was active on the main olive phenolic glycosides, with maximum activity toward oleuropein (100%), followed by ligstroside (65%) and demethyloleuropein (21%). The enzyme showed very low activity with apigenin and luteolin glucosides and was not active on verbascoside and rutin. Kinetic values show that olive beta-glucosidase is 200-fold more active against oleuropein than against the synthetic substrate p-nitrophenyl-beta-d-glucopyranoside (pNPG). According to its catalytic properties, the implication of the purified olive beta-glucosidase on the synthesis of virgin olive oil phenolics is discussed.