The EU White Paper: Energy for the future: Renewable Sources of Energy, COM (97)599, recognizes that there is a need for actions to promote the uptake of liquid biofuels for transport applications. Also, according to the Directive 2003/30/EC of the European Parliament and of the Council of 8 May 2003 on the promotion of the use of biofuels or other renewable fuels for transport, a minimum proportion 2 % of biofuels of all petrol and diesel for transport purposes is encouraged to be achieved by 31 December 2005. This proportion is expected to increase to 5 % by 31 December 2010. Greece is still at an early stage in this field and does not have any commercial activities, as other EU member states (Austria, Germany, France, etc.). Although, two major plants will be build (Central and Northern Greece), until present time there is no biodiesel production in Greece. The production of biodiesel has taken an upward course in the past years. As a result, Europe is now the world's biggest producer and user of biodiesel fuel. This is partly due to European environmental policies aiming at the reduction of carbon dioxide emissions and the implementation of the Directive 2003/30/EC of the European Parliament. Vegetable oils and their derivatives (especially Fatty Acid Methyl Esters) are prominent candidates as alternative diesel fuels. Fatty Acid Methyl Esters (FAMEs), commonly referred to as biodiesel, can be also found from other various renewable resources, such as used frying oils and animal fats. In recent years, increasing attention has been drawn at animal fats and used vegetable oils as sources of biodiesel, primarily as inexpensive feedstocks. In the search of alternative oils for an economic biodiesel production, animal fats should be promoted. In this study, animal fat methyl ester and a commercial automotive diesel fuel were investigated in a stationary single cylinder diesel engine at proportions of 2 % and 5 % respectively. Pure commercial fuel was also used. The fuels were characterized by exposing their major physicochemical properties according to the European Standards EN 590 and EN 14214 and examined under various loads in terms of the exhaust emissions and fuel volumetric consumption. The results proved that the use of animal fat methyl esters provides a significant reduction on particulate matter emissions with respect to the automotive diesel fuel. On the contrary, there was reported an increase in nitrogen oxides (NOx) emissions for the blend containing 5 % animal fat methyl ester and when the engine operated at high loads.