Rhizopus arrhizus lipase (RAL) catalyzed the synthesis of the ester bond between linolenic acid and octanol in a hexane microaqueous system. Lipase activity was determined by measuring increases in absorbance of the hexane at 235 nm, as a function of reaction time, after addition of sufficient silica (0.1 g per 20 mM linolenic acid) to remove free fatty acids, bur not the war product (octyl-linolenate), followed by separation of the silica by centrifugation. The activity of the lipase freeze-dried to the bottom of a glass vial increased linearly as a function of enzyme concentration in the range 0-0.1 EU. RAL displayed maximum activity at pH 7.6 at both water activities of 0.33 and 0.75. Maximum synthetic activity was observed at a water activity of 0.75. The optimum reaction temperature was 40 degrees C, both at pH 7.0 (a(w) = 0.33) and pH 7.6 (a(w) = 0.75). Preliminary kinetic analysis of the synthetic reaction provided an estimate of V-max/K-m for linolenic acid of 0.00095 min(-1) (K-m = 15.2 mM, V-max = 14.6 mu M min(-1) at 50 mM octanol). In comparison, a V-max/K-m of 0.0032 min(-1) (K-m = 12.6 mM, V-max = 40.0 mu M min(-1) at 50 mM octanol) was obtained using a fatty acid-copper salt method for the determination of free fatty acid concentration. No hydrolytic activity could be detected in this system.