A Gram-positive bacterium identified as Nocardia corynebacterioides L29-2 was isolated from a diesel oil contaminated site in southern Taiwan. The bacterium was found able to degrade both straight and branched alkanes in diesel oil. Inorganic nutrients were not able to enhance the growth of the cells but the cell growth and hydrocarbon biodegradation could be accelerated by adding yeast extract. It was found that this bacterium was able to degrade pristane at high efficiency. However, the ability of pristane biodegradation was severely inhibited by the presence of normal alkanes. The diesel oil biodegradation was significantly improved by introducing another bacterium, Rhodococcus globerulus H425-2, found in the same contaminated site. Rhodococcus globerulus H425-2 was found to degrade normal alkanes at a much higher rate than Nocardia corynebacterioides L29-2. In this study, the biodegradation of pristane and n-pentadecane in single substrate and mixed substrate experiments were compared. The cooperation of Nocardia corynebacterioides L29-2 and Rhodococcus globerulus H425-2 was also under investigation. It appeared that the 1: 1 ratio was close to the optimum inoculation ratio of these two species for degrading the mixture of pristane and n-pentadecane at 1: 1 ratio. The results indicate that the addition of organic nutrients and the proper control of the population ratio according to the alkane composition in the contaminated site are crucial for effective bioremediation of diesel spill.