The Centers for Disease Control and Prevention, Atlanta, Ga, recommend use of waterless alcohol hand products in lieu of traditional handwashing for patient care, but there are few data demonstrating the impact of this recommendation on health care-associated infections. To compare the effect of 2 hand hygiene regimens on infection rates and skin condition and microbial counts of nurses' hands in neonatal intensive care units. Clinical trial using a crossover design in 2 neonatal intensive care units in Manhattan, NY, from March 1, 2001, to January 31, 2003, including 2932 neonatal hospital admissions (51 760 patient days) and 119 nurse participants. Two hand hygiene products were tested: a traditional antiseptic handwash and an alcohol hand sanitizer. Each product was used for 11 consecutive months in each neonatal intensive care unit in random order. After adjusting for study site, birth weight, surgery, and follow-up time, there were no significant differences in neonatal infections between the 2 products; odds ratios for alcohol compared with handwashing were 0.98 (95% confidence interval [CI], 0.77-1.25) for any infection, 0.99 (95% CI, 0.77-1.33) for bloodstream infections, 1.61 (95% CI, 0.57-5.54) for pneumonia, 1.78 (95% CI, 0.94-3.37) for skin and soft tissue infections, and 1.26 (95% CI, 0.42-3.76) for central nervous system infections. The skin condition of participating nurses was significantly improved during the alcohol phase (P = .02 and P = .049 for observer and self-assessments, respectively), but there were no significant differences in mean microbial counts on nurses' hands (3.21 and 3.11 log(10) colony-forming units for handwashing and alcohol, respectively; P = .38). Infection rates and microbial counts on nurses' hands were equivalent during handwashing and alcohol phases, and nurses' skin condition was improved using alcohol. However, assessing the impact on infection rates of a single intervention is challenging because of multiple contributory factors such as patient risk, unit design, and staff behavior. Other practices such as frequency and quality of hand hygiene are likely to be as important as product in reducing risk of cross-transmission.