A glycerol-derived biofuel with optimized composition to improve its cold-flow properties was tested in a C class Euro 6b vehicle on a chassis dynamometer following the worldwide harmonized light-duty vehicle test cycle at warm (24 degrees C) and cold (-7 degrees C) ambient conditions. The biofuel was previously blended at 20% v/v with a commercial diesel fuel, which was also tested pure as a reference for comparison. Regulated emissions were measured upstream of the aftertreatment system for an independent evaluation of the effect of the fuel. Previously the equivalence ratio and the exhaust gas recirculation (EGR) rate were analyzed to isolate the effect of these engine parameters from those of the type of fuel. The effect of the fuel used was minor in comparison with the effect of ambient temperature, with cold ambient temperature leading to much higher fuel consumption and gaseous emissions. CO emissions were observed to decrease with the biofuel only at warm ambient conditions, whereas no significant effects of the type of fuel were observed for CO at cold ambient conditions and for total hydrocarbon emissions at any condition. NOx emissions increased slightly at both ambient conditions, which was attributed to a slight decrease in the EGR rate. The main effect was found on particle emissions, which were reduced in both number and mass at both ambient temperatures. No startability problems were observed, even at cold ambient conditions.