Chlorophyllase activity in olive fruit (Olea europaea) was measured using the enzyme solubilized from the protein precipitate. The reaction was stopped by freezing the mixture at -20°C, to avoid dilution of the sample and consequent reduction of the substrate levels to below the detection limits of the analytical system. Separation of the substrates and products of the enzymatic reaction was performed by reverse-phase HPLC using a gradient solvent system of water and ion suppressor/methanol/acetone. These conditions allowed direct resolution of the reaction mixture prior to centrifugation, without the need for the transfer of any of the components to other solvents. Olive chlorophyllase in the crude enzymatic extract showed maximum activity at 50°C and the optimum pH was 8.5 in acetate-phosphate-borate buffer for all substrates used, chlorophylls (a and b) and pheophytins (a and b). The K_m and V_max values obtained for hydrolysis of these substrates showed that chlorophyllase had a greater affinity for chlorophyll b, while the highest maximum rate of reaction occurred with pheophytin a. Substrate inhibition was observed with pheophytin b.