The enzymatic production of biodiesel by methanolysis of cottonseed oil was studied using immobilized Candida antarctica lipase as catalyst in t-butanol solvent. Methyl ester production and triacylglycerol disappearance were followed by HPLC chromatography. It was found, using a batch system, that enzyme inhibition caused by undissolved methanol was eliminated by adding t-butanol to the reaction medium, which also gave a noticeable increase of reaction rate and ester yield. The effect of t-butanol, methanol concentration and temperature on this system was determined. A methanolysis yield of 97% was observed after 24 h at 50 °C with a reaction mixture containing 32.5% t-butanol, 13.5% methanol, 54% oil and 0.017 g enzyme (g oil)−1. With the same mixture, a 95% ester yield was obtained using a one step fixed bed continuous reactor with a flow rate of 9.6 ml h−1 (g enzyme)−1. Experiments with the continuous reactor over 500 h did not show any appreciable decrease in ester yields.