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In spite of the recent trend, voted toward the reduction of renewable energy sources deriving from crops, the EC Commission proposes that the proportion of energy from renewable sources in the transportation sector should be at least 20 % of its final energy consumption by the year 2020. In this framework, the activities aiming to study the effects on engine performances, emissions and fuel consumption of alternative diesel fuel receive continue stimulations and supports. In this paper, results of the different behavior of biodiesel fuels in the injection process and their impact on the air-fuel mixture preparation are reported. The injection process characterization has been carried out in a non-evaporative high-density ambient in order to measure the fuel injection rate and the spatial and temporal distribution of the fuel.