Objectives: The purpose of this research was to demonstrate the effectiveness of hydroperoxide ion-phase transfer catalyst (HPI-PTC) cleaners and disinfectants for maintaining dental unit waterlines free of planktonic organisms. Method and materials: Water samples were taken from 117 sites, which included a variety of dental units and samples from the sink faucets of most operatories. Samples were plated on appropriate bacteriologic media and incubated. The presence or absence of biofilms was confirmed by scanning electron microscopy. Twenty-two of the dental units were retrofitted with independent water systems; the cleaning procedure involved an overnight application of an HPI-PTC cleaner followed by a 2-minute water rinse. Results: Water from both the air-water syringe and the high-speed handpiece lines from all untreated units contained at least 6x10² colony-forming units per milliliter of planktonic or free-floating bacteria; the average was 1.4x10⁵ CFU/mL. An initial 5% solution of HPI-PTC successfully cleared the lines of any apparent biofilm when applied for 3 consecutive days. Thereafter, once weekly use of the cleaner maintained the dental unit water supplies free of significant numbers of planktonic organisms. Conclusion: Routine weekly use of an HPI-PTC cleaner controlled dental unit waterline biofilm and reduced, with minimum effort, the microbial contamination level of water used for patient treatment to less than 200 CFU/mL.