The near-elimination of catheter-related bloodstream infections (CRBSI) in adult intensive care units in Michigan propelled checklists into popular discourse, and stands as one of the landmark achievements of the patient safety field. In this study conducted at all 18 neonatal intensive care units (NICUs) in New York State, use of checklists for central line insertion and maintenance was associated with a marked overall reduction in CRBSI. However, wide variation in infection rates remained between individual NICUs, and hospitals with higher CRBSI rates used the checklists inconsistently. This study demonstrates that effective use of checklists also depends on many local factors, principally the institutional safety culture.