Currently, oil based fuels are the primary energy source of road transport. The growing need for oil independence and CO(2) mitigation has lead to the increasing importance of alternative fuel usage. CO(2) is produced not only as the fuel is used in the vehicle (tank-to-wheel contribution), but also upstream, from the fuel extraction to the refueling station (well-to-tank contribution), and the life cycle of the fuel production (well-to-wheel contribution) must be considered in order to analyse the global impact of the fuel utilization. A road vehicle tank-to-wheel analysis tool that may be integrated with well-to-tank models was developed in the present study. The integration in a demonstration case study allowed to perform a life cycle assessment concerning the utilization of diesel and natural gas fuels in a specific network line of a bus transit company operating in the city of Porto, Portugal.