Aged soils are difficult to treat by biological methods, since contaminants, i.e. hydrocarbon compounds, can be tightly adsorbed into the soil particles. This problem can be solved by using small quantities of specific surfactants to increase the compounds' bioavailability. Selection of the right surfactant and dose is of crucial importance to the biodegradation process, but frequently the selection process is based on a trial-and-error method. Surfactant's hydrophilic-lipophilic balance (HLB) value is an expression of the surfactant's molecule affinity to the organic and water phases. This parameter can be a helpful tool in the selection of the adequate surfactant. The aim of this work is to clarify the combined effect of temperature, and surfactant HLB and dose effects over TPH-diesel removal in a Mexican aged soil. A statistical design was used in order to minimize the number of experiments needed for achieving that purpose. The results of this work indicate that the parameter that mostly affected the biodegradation process was temperature, followed by the surfactant's HLB value, and surprisingly, surfactant dose at the end. On the other hand, TPH-contaminated soil was microbiologically characterized in order to identify the microflora responsible for the biodegradation process. One Bacillus, two Corynebacterium, and three Micrococcus species were found among the existing Gram-positive bacteria. Enterobacter amnigenus, Enterobacter cloacae, and Pseudomonas sp. were detected among the Gram-negative bacteria. (c) 2005 Published by Elsevier Ltd.