The aim of any drinking water supplier is to provide the consumer with potable water of an identical quality of that leaving th treatment plant. However, it has been well documented that water in the consumers' tap is often of inferior microbial quality. Thus there is a need for analytical methods which are quick to perform and reliable in detection of the major groups of pathogens. Bacteria, protozoa and viruses may pass from the source water into the treated drinking water and may become established in biofilm. These attached organisms have been found to be active in absorbing nutrients and more resistant to environmental stresses than free-floating organisms. Analysis of drinking water biofilm are receiving increased attention because biofilm represents the majority of biomass in drinking water and one of the techniques used is FISH. Both DNA and PNA probes have been used along with several methods examining the metabolic activity of the cells, such as DVC (direct viable count), CTC (respiration activity), methods assessing membrane integrity or potential and methods, which indicate enzyme activity, e.g. esterase. Earlier we have shown that E. coli cells with a potential to divide are present in water supplies throughout Europe (Juhna, et al., 2007a) and that E. coli can persist in biofilm for long time, remaining in unculturable form (Juhna, et al., 2007b). Here we report an improvement of PNA FISH protocol where the washing step is eliminated, a study on the passage of active E. coli through the water treatment plant and the role of biofilm in this process. The biofilm was collected at several locations of water treatment plant - raw water source, after coagulation, filtration and in drinking water reservoir - using steel coupons. Results showed that a small number of E. coli may be transported through the water treatment plant but is not detected, probably due to ,,grab sampling" method and culture-based microbial identification methods.