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In an effort to reduce CO2 emissions, governments are increasingly mandating the use of various levels of biofuels. While this is strongly supported in principle within the energy and transportation industries, the impact of these mandates on the transport stock’s CO2 emissions and overall operating efficiency has yet to be fully explored. This paper provides information on studies to assess biodiesel influences and effects on engine performance, driveability, emissions and fuel consumption on state-of-the-art Euro IV compliant Toyota Avensis D4-D vehicles with DPNR aftertreatment systems. Two fuel matrices (Phases 1 & 2) were designed to look at the impact of fuel composition on vehicle operation using a wide range of critical parameters such as cetane number, density, distillation and biofuel (FAME) level and type, which can be found within the current global range of Diesel fuel qualities.