The environmental and social sustainability of biofuel production and use is today the most critical issue for the development of support policies in this sector. The Life Cycle Assessment (LCA) methodology is commonly agreed as the main tool for the estimation of the impact of biofuel chains, even in quantitative terms. This is also reflected in the recently issued EU Directive (Renewable Energy Directive, RED) on the promotion of the use of energy from renewable sources. However, the results of Life Cycle Assessment works largely depend on the quality of the information given as input to the study, as also very recent research works started to investigate: in addition, the comparison of a large number of very different (technically, geographically, agronomically) biofuel chains, as some Life Cycle Assessments and reviews tried to do, is a very difficult task due to the extremely large number of variable conditions and parameters. This paper, by considering a very specific biofuel chain (production and use of Pure/Straight Sunflower Oil in North-Central Italy), discuss some limits and constraints of the application of the LCA method. The work investigated within which boundaries Life Cycle Assessment could be implemented to perform quantitative assessments, as requested by the current supporting policies in the biofuel area. Results showed very large variations in the calculation of the CO2 equivalent emissions, thus illustrating how achievable results depends on the local agricultural practices and performances, even for such a small and well defined biofuel chain. The adoption of the present standardized Life Cycle Assessment approach for generalized evaluations in the bioenergy sector and, in particular, for quantitative assessments should therefore be reconsidered. Concluding, LCA studies, even while addressing very specific and well defined chains, should always provide the bias of the calculations, as this range of variation of Life Cycle Assessment results could be significantly greater than the initially set quantitative targets and therefore the whole investigation would be at risks of inconsistency. Proposals are finally given for small scale projects, with the aim of developing sound but realistic processes to assess biofuel sustainability. (C) 2010 Elsevier Ltd. All rights reserved.