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The exhaust emission and performance characteristics of a 100% biodiesel fuel was evaluated on a single cylinder direct injection diesel engine that had been modified to allow multi-pulse diesel fuel injection at the intake port and independent control of intake heating, exhaust gas recirculation and throttling. Firstly, conventional single-shot direct injection tests were conducted and comparisons made between the use of an ultra-low sulphur diesel fuel and the biodiesel fuel. Secondly, tests for the premixed combustion of neat biodiesel were performed. Exhaust gas recirculation was applied extensively to initiate the low temperature combustion for the conventional in-cylinder single injection operation and to moderate the timing of the homogeneous charge compression ignition for the intake-port sequential injection. Because of the high viscosity and low volatility of the biodiesel, pilot-ignited homogeneous charge compression ignition was used.