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Biodiesel fuels derived from different feedstocks may have significantly different fatty acid profiles and physicochemical properties. To gain further insight into the effect of the biodiesel chemical structure, specifically its degree of unsaturation, on engine performance, combustion characteristics, and emissions, an experimental investigation was conducted on a high-speed direct-injection automotive diesel engine fueled with three mixtures of fatty acid methyl esters. The fuel matrix was designed such that the effect of the degree of unsaturation of the tested biodiesel fuels was isolated. This allowed for the maximization of the effect of the cetane number, while the other properties, such as the chain length, oxygen content, density, viscosity, and volatility, varied within a small range. Results indicated that the degree of unsaturation of biodiesel fuels did not significantly affect engine performance and the start of injection, but it had a noticeable influence on combustion characteristics and emissions, via its effect on the cetane number. A higher degree of unsaturation of biodiesel fuels led to a longer ignition delay and, consequently, a more retarded start of combustion. Regardless of the engine-operating mode, an almost fixed start of injection was attained, while the premixed portion of combustion, peak heat release rate, maximum pressure gradient, peak in-cylinder bulk-gas-averaged temperature, total hydrocarbon (THC) emissions, smoke opacity, and nitrogen oxides (NO x ) emissions increased with the degree of unsaturation.