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These last years, much of researches were carried out to find the appropriate substitution fuel to the fossil fuels. The use of biofuel prepared from non-edible vegetable oils are becoming a promising source to produce a fuel for diesel engine, commonly referred to as “biodiesel”. Considering the high oil extraction yield (around 40%) and the great quantity of pistacia lentiscus (PL) trees available in arid and semi-arid areas of Mediterranean countries, it is selected in the present work to study the biodiesel prepared from PL oil. PL biodiesel is obtained by converting PL seed oil with a single-step homogenous alkali catalyzed transesterification process. PL biodiesel characterization, according to the standard methods, shows that the physicochemical properties are comparable to those of conventional diesel fuel. In a second part, a single cylinder air-cooled, DI diesel engine is used to test PL biodiesel at 1500 rpm under various engine load conditions.