Biodiesel is a renewable fuel which can be used as a direct replacement for fossil Diesel fuel as a calorific source in Diesel Engines. It consists of fatty acid mono-alkyl esters, which are produced by the trans-esterification reaction of plant oils with monohydric alcohols. The Plant oils and alcohols can both be derived from biomass, giving this fuel the potential for a sustainable carbon dioxide neutral life-cycle, which is an important quality with regard to avoiding the net emission of anthropogenic greenhouse gases. Depending on its fatty ester composition, Biodiesel can have varying physical and chemical properties which influence its combustion behaviour in a Diesel engine. It has been observed by many researchers that Biodiesel can sometimes lead to an increase in emissions of oxides of nitrogen (NOx) compared to fossil Diesel fuel, while emitting a lower amount of particulate mass. The work described in this paper examines the influence of the detailed molecular structure of fatty acid ester molecules on the formation of NOx and particulate matter. Several individual fatty acid alcohol esters were synthesised and tested as fuel in a single-cylinder direct injection Diesel engine under carefully controlled operating conditions. Cylinder pressure and exhaust gas emissions were measured and exhaust particulate number and size distribution were recorded using a differential mobility spectrometer. An ignition improving additive was used in certain experiments to eliminate the influence of ignition delay on the combustion characteristics of the various molecules. It was observed that the chain length and the degree of saturation of the fatty acids, as well as the type of alcohol used for the fuel synthesis, have distinct effects on the formation of NOx and particulate matter during Diesel combustion.