The hydrolytic activity of a partially purified chlorophyllase, obtained from the alga Phaeodactylum tricornutum, was determined in a biphasic organic system using different mixtures of hexane and Tris-HCl buffer solution (20 mmol dm-3, pH 7.5). The most appropriate kinetic parameters were determined for the optimum hydrolytic activity of chlorophyllase including hexane concentration (45%), optimum pH (7.5), time of incubation (7 h), incubation temperature (25-degrees-C) and enzyme concentration (10 mug cm-3). The optimum concentrations of magnesium chloride and dithiothreitol, used as activators, were determined to be 4 mmol dm-3 and 5 mmol dm-3, respectively. The results also indicated that diisopropyl fluorophosphate (DIFP) had a mixed competitive non-competitive inhibitory effect on chlorophyllase activity. The end-products of the chlorophyllase hydrolytic reaction were identified and confirmed, using thin-layer and high-performance liquid chromatographies, spectrophotometric scanning and Fourier transform infrared spectroscopy.