The effects of traditional biodiesel (fatty acid methyl-esters, FAME) and a hydrotreated vegetable oil (HVO) were comprehensively investigated on a production Euro 6 diesel car, including fuel injection rate and timing, combustion analysis, emissions of regulated and unregulated pollutants, and regeneration of the diesel particle filter. The use of both biofuels is a part of the efforts to reduce emissions of greenhouse gases and health-relevant pollutants and to improve energy security and sustainability. HVO, albeit more expensive, offers benefits relative to FAME in terms of oxidation stability, injector fouling, energy content and cetane number. The car was fitted with an on-board instrumentation and subjected to a range of driving cycles on a chassis dynamometer. The fuel consumption calculated from instantaneous emissions data based on exhaust gas composition measured by an on-board FTIR and calculated exhaust flow matched directly measured fuel consumption within several percent on all fuels; differences in the consumption among the fuels correspond to different heating values. The combustion onset and maximum heat release rate were comparable for diesel and FAME but were advanced on HVO due to its higher cetane number, causing, at times, multiple distinct heat release peaks, suggesting that optimization of fuel injection timing for HVO might be beneficial. Emissions of methane and ammonia were negligible, of NO were measurable and slightly lower for HVO than for other fuels, of formaldehyde were limited to cold engine accelerations and highest for FAME and negligible for HVO, of NO and NO were high on all fuels during all operating conditions except for the type approval test. The results confirm several relative advantages of HVO over RME, with penetration into engine lubricating oil during particle filter regeneration to be further investigated. The effects of HVO lubricity and other long-term effects were not evaluated here.