Plug-in hybrid electric vehicles (PHEVs) could reduce transportation air emissions and energy use. However, a method is needed for estimating on-road emissions of PHEVs. To develop a framework for quantifying microscale energy use and emissions (EU&E), measurements were conducted on a Toyota Prius retrofitted with a plug-in battery system on eight routes. Measurements were made using the following: (1) a data logger for the hybrid control system; (2) a portable emissions measurement system; and (3) a global positioning system with barometric altimeter. Trends in EU&E are estimated based on vehicle specific power. Energy economy is quantified based on gasoline consumed by the engine and grid energy consumed by the plug-in battery. Emissions from electricity consumption are estimated based on the power generation mix. Fuel use is approximately 30% lower during plug-in battery use. Grid emissions were higher for CO₂, NO(x), SO₂, and PM compared to tailpipe emissions but lower for CO and hydrocarbons. EU&E depends on engine and plug-in battery operation. The use of two energy sources must be addressed in characterizing fuel economy; overall energy economy is 11% lower if including grid energy use than accounting only for fuel consumption.