BACKGROUND: Health care-associated infections most commonly result from person-to-person transmission via the hands of health care workers. METHODS: We studied the efficacy of hand hygiene agents (n = 14) following 10-second applications to reduce the level of challenge organisms (Serratia marcescens and MS2 bacteriophage) from the hands of healthy volunteers using the ASTM-E-1174-94 test method. RESULTS: The highest log 10 reductions of S marcescens were achieved with agents containing chlorhexidine gluconate (CHG), triclosan, benzethonium chloride, and the controls, tap water alone and nonantimicrobial soap and water (episode 1 of hand hygiene, 1.60-2.01; episode 10, 1.60-3.63). Handwipes but not alcohol-based handrubs were significantly inferior from these agents after a single episode of hand hygiene, but both groups were significantly inferior after 10 episodes. After a single episode of hand hygiene, alcohol/silver iodide, CHG, triclosan, and benzethonium chloride were similar to the controls in reduction of MS2, but, in general, handwipes and alcohol-based handrubs showed significantly lower efficacy. After 10 episodes, only benzethonium chloride (1.33) performed as well as the controls (1.59-1.89) in the reduction of MS2. CONCLUSIONS: Antimicrobial handwashing agents were the most efficacious in bacterial removal, whereas waterless agents showed variable efficacy. Alcohol-based handrubs compared with other products demonstrated better efficacy after a single episode of hand hygiene than after 10 episodes. Effective hand hygiene for high levels of viral contamination with a nonenveloped virus was best achieved by physical removal with a nonantimicrobial soap or tap water alone.