The target of the European Union (EU) from the 1990s has been to reduce the level of greenhouse gas (GHG) in the climate by 40 % by 2030 [1]. Currently the transport sector is one of the biggest greenhouse gas emission producer in the EU [2]. Drop-in biofuels can contribute to the reduction of GHG emissions in the transport sector. Diesel R33, a newly developed biofuel enables sustainable mobility fulfilling the European diesel fuel specification and reduces the GHG emissions by about 18.2 % against fossil diesel fuel. Diesel R33 is made of 7 % used cooking oil methyl ester, 26 % hydrotreated vegetable oil (HVO) and 67 % high quality diesel fuel. HVO was produced from rapeseed and palm oil. This new biofuel was tested in a fleet of 280 vehicles (passenger cars, light duty vehicles, off-road vehicles and urban buses) covering all emission classes. The impact of the new fuel on the vehicles, their emissions and the engine oil aging was investigated. Additional to the extended fleet test, thermo-oxidative laboratory aging tests using Diesel R33 in non-additivated base oil, in squalane as model substance for base oil and in real engine oil were carried out systematically to determine the impact of Diesel R33 on oil sludge formation. Moreover, five passenger cars (emission classes Euro 3, Euro 5 and Euro 6) were intensively tested on exhaust gas emissions of regulated and non-regulated components. The results showed all vehicles fulfilled the exhaust gas regulation on running on CRC reference diesel fuel as well as on Diesel R33. HC and CO were reduced with Diesel R33 but NOx emissions were slightly increased. Non-regulated emissions such as carbonyls were found to have reduced. The mutagenicity of the exhaust gases can be reduced by using Diesel R33. These positive impacts were clearly observed only with the Euro 3 passenger car. Due to the overall low emission level, Euro 6 cars showed no significant changes in the non-regulated emissions or health effects.