An experimental campaign has been devoted to characterize the engine fuelled by the ethanol based blend highlighting the advantages and issues related to the bio-ethanol use. The effects of the most important injection settings on the engine performance have been detailed, applying a Design of Experiment (DoE) method, to identify the most important parameters affecting emissions and the potentiality offered by a proper engine calibration to optimize the ethanol blend use. The tests were performed on a 2.0 L Euro 5 diesel engine, in steady-state at partial and full-load conditions, burning two fuel blends: a Rapeseed Methyl Ester (RME)/diesel blend with 10% of biodiesel by volume, assumed as reference fuel, and a bio-ethanol/RME/Diesel with 20% of bio-ethanol and 10% of biodiesel by volume. Emission measurements with standard Euro 5 calibration showed the well-know effects of ethanol blending to diesel fuel: a strong smoke emissions reduction in all tested conditions, together with a decrease of NOx exhaust concentration. An increment of CO and HCs emissions for bio-ethanol blends were also found, mainly at low load conditions. This issue has been investigated performing a specific test campaign inspired to the DoE method. Such activity identified in the calibration of the pilot injection quantity and rail pressure values the most influential factors in the gaseous unburned reduction. By the adoption of an optimized calibration, identified by DoE results, gaseous emissions on NEDC cycle were significantly reduced with respect to the reference calibration, matching the HCs and CO Euro 5 limits and approaching both PM and NOx Euro 6 emission levels. (C) 2013 Elsevier Ltd. All rights reserved.