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The dissertation provides the basic requirements for alternative fuels with regard to their application as fuels for internal combustion engines, and it analyzes the characteristics of vegetable oils as potential fuels for diesel engines and the possibilities of adapting them to the requirements of modern diesel engines, with a particular focus on rapeseed oil. The thesis includes a review (with basic characteristics) of the raw material base for obtaining, first vegetable oils, and then biodiesel (vegetable oil methyl or ethyl-ester), as well as an overview of goals, the current state and trends in the previous period in the European Union concerning the ratio of raw material bases in biodiesel production, the consumption, production, import and supply of biodiesel, and a plan for future biodiesel exploitation in the Republic of Serbia. In the process of transformation of the chemical energy of fuel into the mechanical work in diesel engines, the manner and quality of fuel-air mixture formation has a dominant influence, which is in turn highly affected by the fuel injection system. The thesis analyzes the operation of a selected fuel injection system with the aim of identifying and observing the importance and effects of certain physical characteristics of fuel on the operation of the fuel injection system, which emphasizes the significance of the knowledge of the speed of sound, density and fuel bulk modulus. To determine the dependence on the speed of sound, density and bulk modulus of selected fuels (UR, B100, B50 and diesel) on the pressure (ranging from the atmospheric to 1600 bar), an original experimental method was conceived and a purpose-built apparatus was installed. After ascertaining the above physical characteristics of examined fuels, the operation characteristics of selected fuel injection systems were determined experimentally for three different fuels (B100, B50 and diesel). The results imply the influence of the differences in the speed of sound, bulk modulus and density of selected fuels on the characteristics of the fuel injection system, meaning that when biodiesel is used the pressure in front of the injector starts to increase earlier and fuel injection engages earlier as well, the injection pressure reaches its maximum earlier, a higher injection pressure is obtained, the injection time is longer and the fuelling is higher, compared to the operation with diesel. A numerical simulation was employed to determine the injection’s characteristic and the integral injection’s characteristic. Also simulated were the fuel pressure curves and the injector needle lift curves, with the implementation of mathematical dependences for determining the physical characteristics of fuels, obtained on the basis of experimental data. The visualization of the injected fuel spray showed that the biodiesel spray is narrower, the spray length is longer and the spray projected area is smaller in comparison with diesel.