The objective of this research is to evaluate differences in fuel consumption and tailpipe emissions of flexible fuel vehicles (FFVs) operated on ethanol 85 (E85) versus gasoline. Theoretical ratios of fuel consumption and carbon dioxide (CO2) emissions for both fuels are estimated based on the same amount of energy released. Second-by-second fuel consumption and emissions from one FFV Ford Focus fueled with E85 and gasoline were measured under real-world traffic conditions in Lisbon, Portugal, using a portable emissions measurement system (PEMS). Cycle average dynamometer fuel consumption and emission test results for FFVs are available from the U.S. Department of Energy, and emissions certification test results for ethanol-fueled vehicles are available from the U.S. Environmental Protection Agency. On the basis of the PEMS data, vehicle-specific power (VSP)-based modal average fuel and emission rates for both fuels are estimated. For E85 versus gasoline, empirical ratios of fuel consumption and CO2 emissions agree within a margin of error to the theoretical expectations. Carbon monoxide (CO) emissions were found to be typically lower. From the PEMS data, nitric oxide (NO) emissions associated with some higher VSP modes are higher for E85. From the dynamometer and certification data, average hydrocarbon (HC) and nitrogen oxides (NOx) emission differences vary depending on the vehicle. The differences of average E85 versus gasoline emission rates for all vehicle models are -22% for CO, 12% for HC, and -8% for NOx emissions, which imply that replacing gasoline with E85 reduces CO emissions, may moderately decrease NOx tailpipe emissions, and may increase HC tailpipe emissions. On a fuel life cycle basis for corn-based ethanol versus gasoline, CO emissions are estimated to decrease by 18%. Life-cycle total and fossil CO2 emissions are estimated to decrease by 25 and 50%, respectively; however, life-cycle HC and NOx emissions are estimated to increase by 18 and 82%, respectively.