Hand hygiene is an effective means of preventing hospital-associated infection, but compliance among health care workers is poor. Few studies aimed at increasing hand hygiene in the hospital setting have shown sustained improvement and concurrent decreases in hospital-associated infections, and even fewer have been performed in the pediatric setting. We implemented a hand hygiene program with the hopes of improving hand hygiene and decreasing hospital-associated rotavirus infection rates. A multidisciplinary group developed a hospital supported, house-wide campaign. Opportunities for hand hygiene were observed during 5 periods. The frequency of hospital-associated rotavirus infection was tracked over time by review of laboratory records. Correlates of hand hygiene were investigated with the use of multivariate logistic regression. Overall hand hygiene compliance improved from 62% in period 1 to 81% in period 5 (P < 0.001). Soap and water was the most common method for practicing hand hygiene, and alcohol hand gel use increased from 4% to 29% between the first and last observation periods (P < 0.001). The rate of hospital-associated rotavirus infection decreased from 5.9 episodes per 1000 discharged patients in 2001 to 2.2 episodes per 1000 discharged patients in 2004 (P = 0.01). Period of observation, hospital ward, type of care provider and type of care performed were all independently associated with hand hygiene (adjusted P < or = 0.02 for all). Improving hand hygiene is an important goal for health care institutions. These data can be useful for development of interventions aimed at improving hand hygiene.