Potential improvement on exhaust emissions, biodegradability and the possibility to reduce dependence on fossil fuel resources has led to an increasing interest on the use of biofuels for transport application. In this work, the analysis of the spray behaviour of first and second generation biodiesel in a Euro 5, common rail transparent diesel engine has been performed. GTL, SME and RME fuels have been used in blends at 100% and 50% in volume; while reference fuel consisted of commercial diesel. Two engine operating conditions of the NEDC have been selected: 1500 rpm at 2 bar of brake mean effective pressure (BMEP) and 2000 rpm at 5 bar BMEP. The injection process has been accurately studied, and the influence of the combustion process on the spray behaviour has been taken into account. Typical jets parameters such as penetration and cone angles have been detected and a comparison with theoretical models of Hiroyasu and Siebers has been performed. A new correlation for the forecasting of the jet penetration has been obtained starting from Hiroyasu equations. An image-based method has been applied for the identification of the phenomena that control the spray behaviour during its evolution in the combustion chamber. First generation biodiesels, pure and blends, show longer penetration with respect to the reference fuel at both the engine speed analysed. Moreover, they penetrate for a longer time in the combustion chamber, because of the longer energizing time set, so impingement phenomena can be observed. On the other hand, the second generation biodiesels penetrate less than reference one, due to its lower density, but also because the combustion of the pilot injection causes an increase of pressure that obstructs the penetration in the combustion chamber. Finally, a good agreement between the breakup times computed by means of the Hiroyasu and Siebers correlations and the ones from the experimental data has been found. (C) 2011 Elsevier Ltd. All rights reserved.