Lipase from Candida rugosa was immobilized onto chitosan using four different protocols. The variation of crystallinity (5.57-92.86%), which was a result of thermal treatments and crosslinking of the chitosan, influenced the protein load (7.46-25.15 mg g(-1) chitosan) and protein load efficiency (21.67-41.68%) for immobilization assays made with identical lipase solution concentration (1.3 mg of protein/mL). The effects of protein load (10, 30, 50 and 70 mg of lipase), reaction temperature (30, 40, 50, 60, 70 A degrees C) and substrates molar ratio (0.05-0.30 M) have been studied in the butyl oleate synthesis in iso-octane when water activity of the free and immobilized enzymes were fixed around 0.53 +/- A 0.04. The catalytic activity of the immobilized lipase has also been tested. The Ping-Pong bi-bi mechanism with dead end complex of n-butanol was found to fit the initial rate data. The values of the apparent kinetic parameters were determined by graphic and parametric method as: V (max) = 18.2-19.0 mmol min(-1) g(-1); K (M; Acid) = 0.599-0.640 mol L-1; K (M; Alcohol) = 0.128-0.149 mol L-1; and K (i; Alcohol) = 1.933 mol L-1.