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Biofuels development and specification are currently driven by the engine (mainly gasoline- and diesel-type) technology, existing fossil fuel specification and availability of feedstock. The ability to use biofuels with conventional fuels without jeopardising the standard fuel specifications is a very effective means for the implementation of these fuels. In this work the effect of dual fuelling with in-cylinder injected ULSD fuel or synthetic second generation biofuels (a Gas-To-Liquid GTL fuel as a surrogate of these biofuels as its composition, specifications and production process are very similar to second generation biofuels) and with inlet port injected bioethanol on the engine performance and emissions were investigated. The introduction of anhydrous bioethanol improved the NOx and smoke emissions, but increased total hydrocarbons and carbon monoxide.