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Diesel fuel contamination in soils may be toxic to soil microorganisms and plants and acts as a source of groundwater contamination. The objective of this study was to evaluate the soil biological activity and phytotoxicity to garden cress (Lepidium sativum L.) in a soil polluted with diesel fuel. For this, a diesel fuel spill was simulated on agricultural soil at dose 11 m(-2). During the experiment (400 days) the soil was not covered in vegetation and no agricultural tasks were carried out. A stress period of 18 days following the spill led to a decrease in soil biological activity. reflected by the soil microbial biomass and soil enzymatic activities, after which it increased again. The n-C-17/Pristine and n-C-18/Phytane ratios were correlated negatively and significantly with the dehydrogenase. arylsulphatase, protease, phosphatase and urease activities and with the soil microbial biomass during the course of the experiment. The beta-glucosidase activity indicated no significant connection with the parameters related with the evolution of hydrocarbons in the soil. Finally, the germination activity of the soil was seen to recover 200 days after the spill. (C) 2009 Elsevier B.V. All rights reserved.