OBJECTIVES: Hand hygiene promotion interventions rarely result in sustained improvement, and an assessment of their impact on individual infection risk has been lacking. We sought to measure the impact of hand hygiene promotion on health care worker compliance and health care-associated infection risk among neonates. METHODS: We conducted an intervention study with a 9-month follow-up among all of the health care workers at the neonatal unit of the Children's Hospital, University of Geneva Hospitals, between March 2001 and February 2004. A multifaceted hand hygiene education program was introduced with compliance assessed during successive observational surveys. Health care-associated infections were prospectively monitored, and genotypic relatedness of bloodstream pathogens was assessed by pulsed-field gel electrophoresis. A comparison of observed hand hygiene compliance and infection rates before, during, and after the intervention was conducted. RESULTS: A total of 5325 opportunities for hand hygiene were observed. Overall compliance improved gradually from 42% to 55% across study phases. This trend remained significant after adjustment for possible confounders and paralleled the measured increase in hand-rub consumption (from 66.6 to 89.2 L per 1000 patient-days). A 9-month follow-up survey showed sustained improvement in compliance (54%), notably with direct patient contact (49% at baseline vs 64% at follow-up). Improved compliance was independently associated with infection risk reduction among very low birth weight neonates. Bacteremia caused by clonally related pathogens markedly decreased after the intervention. CONCLUSIONS: Hand hygiene promotion, guided by health care workers' perceptions, identification of the dynamics of bacterial contamination of health care workers' hands, and performance feedback, is effective in sustaining compliance improvement and is independently associated with infection risk reduction among high-risk neonates.