Renewable and bio-based transportation fuel sources can lower the life-cycle greenhouse gas emissions from vehicles. Here, we present an initial assessment of ethyl 3-ethoxybutyrate (EEB) as a biofuel in terms of its performance as a fuel oxygenate and its persistence in the environment. EEB can be produced from ethanol and poly-3-hydroxybutyrate, a bacterial storage polymer that can be produced from non-food biomass and other organic feedstocks. The physicochemical properties of EEB and fuel-relevant properties of EEB-gasoline blends were measured, emissions of criteria pollutants from EEB as a gasoline additive in a production vehicle were evaluated, and fate and persistence of EEB in the environment were estimated. EEB solubility in water was 25.8 g/L, its K<
sub>
ow<
/sub>
was 1.8, and its Henry's Law constant was 1.04 x 10<
sup>
-5<
/sup>
atm-m<
sup>
3<
/sup>
/mole. The anti-knock index values for 5% and 20% v/v EEB-gasoline blends were 91.6 and 91.9, respectively. Reductions in fuel economy were consistent with the level of oxygenation, and criteria emissions were met by the vehicle operated over the urban dynamometer driving cycle (FTP 75). Predicted environmental persistence ranged from 15 d to 30 d which indicates that EEB is not likely to be a persistent organic pollutant. Combined, these results suggest a high potential for the use of EEB as a renewable fuel source.