Limiting factors induced by the formation of a biofilm on the hydrocarbon's surface during in-situ bioremediation were identified. A liquid-liquid system (hydrocarbon-aqueous solution) and a device made of six fixed-bed columns packed with sand were successively used on a laboratory-scale. In the liquid-liquid system, it was shown that biofilm growth was limited by the diffusion of both oxygen and hydrocarbon within the biofilm. At the end of growth, the biofilm thickness reached 80 +/- 20mum. In the fixed-bed columns, additional difficulties were observed. First, distribution of oxygen in the deepest part of the sand was difficult: from 84 to 100% of the biofilm growth occurred in the upper two-thirds of the sand. Then, the surface of the hydrocarbon available for microorganisms was found to be a limiting factor for the biofilm's development. (C) 2004 Society of Chemical Industry.