Our knowledge of the microbial characteristics of deep subsurface waters is currently very limited, mainly because of the methods used to collect representative microbial samples from such environments. In order to improve this procedure, a protocol designed to remove the unspecific, contaminant biofilm present on the walls of an approximately 800 m deep well is proposed. This procedure included extensive purges of the well, a mechanical cleaning of its wall, and three successive chlorine injections to disinfect the whole line before sampling. Total bacterial counts in water samples decreased from 2.5 x 10(5) to 1.0 x 10(4) per millilitre during the cleaning procedure. Culture experiments showed that the first samples were dominated by sulfate-reducers and heterotrophs, whereas the final sample was dominated by oligotrophic and hydrogenotrophic bacteria. Community structures established on the diversity of the 16S rRNA genes and data analysis revealed that the water sample collected, after a purge without removal of the biofilm, was characterized by numerous phyla which are not representative of the deep subsurface water. On the other hand, several bacterial phyla were only detected after the full cleaning of the well, and were considered as important components of the subsurface ecosystem which would have been missed in the absence of well cleaning.