Water from dental units (DU), used for cooling and clearing the field of dental operations, is frequently contaminated by microorganisms. Retrograde spread of oral microbes into DU tubuing, contaminated plumbing systems and endogenous DU contamination have been implicated. This study investigated the contribution of DU tubing to this contamination in 11 randomly selected DU. The times required, under standardized conditions, for DU bacterial levels to decrease in response to the flushing caused by DU operation, or increase in response to stagnation caused by shutting down the DU, were measured. The DU tubing was then removed and similarly manipulated. The results showed similar bacterial levels and populations in the DU and their corresponding tubes. Sixteen control samples taken from the connecting plumbing system at distant locations, after periods of stagnation which result in DU bacterial contamination, were negative. This suggests the plumbing, in our system, is not an important factor. Thus, DU can endogenously contaminate the water passing through them; their tubes have the potential to generate similar magnitudes of bacterial contamination to that determined from intact DU. Scanning electron microscopy of the tube lumens showed a biofilm, characterized by microorganisms embedded in an amorphous matrix in all cases. This biofilm could act as a reservoir to facilitate rapid recontamination. Further analysis of the data indicates there could be other contributing factors.