Background: The objective of this study was to evaluate the efficacy of the cleaning and bacterial killing ability of a new non–enzyme-based formulation (killing detergent solution [KDS]) compared with commercially available enzymatic detergents that included Metrizyme (Metrex Research Division of Sybron Canada Ltd. Morrisburg, Ontario) and Gzyme (Germiphene Corp, Brantford, Ontario). KDS is a hydrogen peroxide–based detergent formulation that combines cleaning efficacy with the ability to kill microorganisms. The KDS formulation helps ensure the protection of the health care worker from infectious risk during the soaking and cleaning stages of medical device reprocessing and reduces the bioburden on devices before sterilization/disinfection. Methods: Test organisms that included Enterococcus faecalis, Salmonella choleraesuis, Staphylococcus aureus, and Pseudomonas aeruginosa were suspended in artificial test soil (ATS-B; patent submitted), inoculated at 10⁶ colonyforming units per carrier and dried overnight before detergent exposure. The ATS-B mimics the blood, protein, carbohydrate, and endotoxin levels of patient-used medical devices. Plastic lumen carriers and a flexible colonoscope were used for surface and simulated-use testing, respectively. Results: The results for the microbial challenge dried onto polyvinyl chloride (PVC) carriers demonstrated that the ability of KDS to remove protein, blood, carbohydrate, and endotoxin from surface test carriers was as effective as the enzyme detergents that were evaluated. Furthermore, KDS was able to effect approximately a 5-Log₁₀ reduction in microbial loads with a 3-minute exposure at room temperature, whereas none of the other detergents were as effective. In simulated-use testing of a soiled colonoscope, KDS was significantly better at ensuring microbial killing compared with Gzyme and Metrizyme and was equivalent to the enzymatic detergents in cleaning ability. Conclusions: In summary the KDS has excellent microbial-killing ability in 3-minute exposures at room temperature and cleans as well as the existing enzymatic detergent formulations that were tested. (Am J Infect Control 2001;29:168-77)