The use of hydrogen produced from renewable energy sources is often reported as an attractive strategy to address the issue of urban environmental sustainability in automotive sector, and a potential alternative to fossil fuel-fed vehicles. The project ‘Filiera Idrogeno’ (Hydrogen Chain) has investigated the potential realisation of hydrogen production chains from renewables and its use as automotive fuel in Tuscany Region (Italy). In this context, life cycle assessment was used for evaluating the environmental sustainability of such chains, applied to a fleet of hydrogen vehicles for urban commercial delivery. From the energy supply side, renewable wind and biomass energy sources were considered for hydrogen production either by electrolysis or direct separation from biomass gasification syngas, according to specific simulations. Benchmarking with hydrogen produced by using Italian electricity mix was carried out. From the transport side, vehicles equipped with either fuel cell or internal combustion engine were evaluated. A benchmarking analysis with standard electric vehicles supplied with electricity produced from the same renewable energy sources was also carried out. The results give some indications on the environmental aspects of the different alternatives and on the contribution of the chain phases to the overall impacts.