Background. Infections cause morbidity and mortality in neonatal intensive care units (NICUs). The association between nursery design and nosocomial infections is unclear. objective. To determine whether rates of colonization by methicillin-resistant Staphylococcus aureus (MRSA), late-onset sepsis, and mortality are reduced in single-patient rooms. design. Retrospective cohort study. setting. NICU in a tertiary referral center. methods. Our NICU is organized into single-patient and open-unit rooms. Clinical data sets including bed location and microbiology results were examined over 29 months. Differences in outcomes between bed configurations were determined by χ2 and Cox regression. patients. All NICU patients. results. Among 1,823 patients representing 55,166 patient-days, single-patient and open-unit models had similar incidences of MRSA colonization and MRSA colonization-free survival times. Average daily census was associated with MRSA colonization rates only in singlepatient rooms (hazard ratio, 1.31; P =.039), whereas hand hygiene compliance on room entry and exit was associated with lower colonization rates independent of bed configuration (hazard ratios, 0.834 and 0.719 per 1% higher compliance, respectively). Late-onset sepsis rates were similar in single-patient and open-unit models as were sepsis-free survival and the combined outcome of sepsis or death. After controlling for demographic, clinical, and unit-based variables, multivariate Cox regression demonstrated that bed configuration had no effect on MRSA colonization, late-onset sepsis, or mortality. conclusions. MRSA colonization rate was impacted by hand hygiene compliance, regardless of room configuration, whereas average daily census affected only infants in single-patient rooms. Single-patient rooms did not reduce the rates of MRSA colonization, late-onset sepsis, or death.