Environmental concerns related to intensive fossil fuel use have pursued the development of programs to promote the production and use of renewable fuels. This is specially true for diesel fuel used in transit bus fleets, in which the intensive and concentrated use in densely populated areas generates adverse environmental effects in cities all over the world. Although improvements in fossil fuel quality have been achieved in the last decades, most have to be done to make breath cleaner in world metropolis. One of the most promising options, known as Biodiesel, is an ester produced from vegetable oil or animal fat, in a chemical reaction with methanol or ethanol, through a process known as transesterification. It contains, on average, 8% and 12.5% less energy than diesel fuel in a volume and in a weight basis, respectively, which affects engine power, in accordance with the blend used. Biodiesel and, hence, biodiesel blends, as oxygenated fuels, may reduce engine's emissions, like smoke, PM, CO and HC. However, oxygen content also increases NOx emissions, which has to be considered for ozone nonattainment areas. As a biofuel, it degrades with the presence of water, oxygen and elevated temperatures, forming products that can be detrimental to engine components, and also can freeze or become a gel into the injection system at low temperatures, which demands a strict control of its properties, and blend percentages, in accordance with technical standards established by regulatory authorities. Brazil, one of the world leaders in Biodiesel use, with a compulsory program of 5% blend for all the country's diesel fleet, had an annual consumption of 2.5 billion of litters in 2011, 80% of which derived from soybean. Besides the regular 5% blend program, there are some experimental programs with transit bus fleets, ranging from 20% to 100% blends, with a high mileage and technical experience. This knowledge, allied to available capacity of Brazilian biodiesel industry, has enforced technical discussions about standard improvements and the increasing of biodiesel blend use for transit bus fleets in metropolitan areas. Also, the so called “Sugarcane Diesel”, obtained from sugarcane thought a “biorefining” process, and used in a 10% blend in diesel engines without any modifications in experimental programs with transit fleets in São Paulo and Rio de Janeiro, has showed PM emission improvements, without any increase in fuel consumption and NOx emissions. This work is supposed to present an overview of biodiesel engine technology and standardization process and some of the results obtained in Brazilian Transit Bus Fleets Experiments, highlighting the possible next steps, into a technical and regulatory perspective.