Microbial contamination of dental unit waterlines is thought to be the result of biofilm formation within the small-bore tubing used for these conduits. Systematic sampling of 121 dental units located at the dental school of Université de Montréal showed that none of the waterlines was spared from bacterial contamination. Multilevel statistical analyses showed significant differences between samples taken at the beginning of the day and samples taken after a 2-min purge. Differences were also found between water from the turbine and the air/water syringe. Random variation occurred mainly between measurements (80%) and to a lesser extent between dental units (20%). In other analyses, it was observed to take less than 5 days before initial bacterial counts reached a plateau of 2 x 10(5) CFU/ml in newly installed waterlines. Sphyngomonas paucimobilis, Acinetobacter calcoaceticus, Methylobacterium mesophilicum, and Pseudomonas aeruginosa were the predominant isolates. P. aeruginosa showed a nonrandom distribution in dental unit waterlines, since 89.5% of the all the isolates were located in only three of the nine clinics tested. Dental units contaminated by P. aeruginosa showed significantly higher total bacterial counts than the others. By comparison, P. aeruginosa was never isolated in tap water remote from or near the contaminated dental unit waterlines. In conclusion, dental unit waterlines should be considered an aquatic ecosystem in which opportunistic pathogens successfully colonize synthetic surfaces, increasing the concentration of the pathogens in water to potentially dangerous levels. The clinical significance of these findings in relation to routine dental procedures is discussed.