Using bioalcohols as a renewable energy source to substitute a part of fossil energy traffic and increasing the sustainability of individual transportation are important objectives in several countries. The global share of Bioethanol used for transportation is continuously increasing. Ethanol is a biomass-based renewable fuel that can be produced by alcoholic fermentation of sugar beet, sugar can, corn, and wheat (bio-ethanol), although petro-ethanol also exists, i.e., ethanol produced from fossil fuels. In the present tests we performed repeated cold starts performed with all investigated fuels, in two temperature ranges approaching 0ºC and 20ºC and with online measurement of different legislated and non-legislated emission components. The investigated fuel contained ethanol (E), in the portions of 10% and 85% by volume. The investigated vehicle represented a newer state of technology and an emission level of Euro 5. The engine works with homogenous GDI concept and with 3-W-catalyst (3WC). Since there is a special concern about the particle emissions of gasoline cars with direct injection, the nanoparticle counts measurements were systematically performed. The non-legislated gaseous emissions were tested at the tailpipe with FTIR, this with special focus on NH₃, HCHO (formaldehyde), and MeCHO (acetaldehyde).