Abstract The intracellular lipase produced by the fungus Rhizopus oryzae was studied in this report. Optimum conditions of lipase activity were determined. This lipase was most active at pH 8.5 and the optimum temperature was observed at 30°C. Optimization of the medium conditions was carried out with a fractional design in two steps. Lipolytic activity, after optimization, was greater than the one which was determined from the initial medium. The decrease in lipase activity of R. oryzae mycelia was studied in alkanes, acetone, ethers and chloroalkanes. The kinetic model obtained showed that the decrease in activity followed a succession of two first-order reactions. R. oryzae mycelium was more stable in hydrophobic solvents.