BACKGROUND The maximum recommended level of microbial contamination of water from dental unit waterlines (DUWL) is 200 colony-forming units per milliliter (CFU/mL). This article addresses the importance of water selection in achieving that standard. METHODS Microbial contamination in water samples from 75 new dental units, with a closed-circuit water system, were compared using combinations of tap water and sterile distilled water with and without two chemical disinfectants (bleach and 0.12% chlorhexidine gluconate, Bio2000) over a six-week period. Baseline tap water samples were collected and tested initially. RESULTS The microbial plate counts of seven tap water specimens (controls) ranged from 4 to 95 CFU/mL. These results were well below both the 500 CFU/mL standard for public drinking water and the 200 CFU/mL goal for dental treatment water. However, when passed through dental units, no significant bacterial reduction was achieved for samples of tap water (Group 1), tap water treated with bleach (Group 2), or tap water treated with Bio2000 (Group 4). Only water samples from dental units using Bio2000 alone (Group 3) or a combination of sterile, distilled water with Bio2000 (Group 5) met or exceeded the 200 CFU/mL standard. CONCLUSIONS Using tap water alone or tap water with bleach did not improve water quality. However, the American Dental Association (ADA) standard for reduced microbial contamination of dental unit waterlines was met using Bio2000 and distilled water treated with Bio2000. CONCLUSIONS The ADA standard of 200 CFU/mL was achieved using a closed water system and distilled water treated with Bio2000. Using 100% Bio2000 is also effective, but more costly.