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background Variation in health care delivery and outcomes in NICUs may be partly explained by differences in safety culture. objective To describe NICU caregiver assessments of safety culture, explore the variability within and between NICUs on safety culture domains, and test for association with caregiver characteristics. methods We surveyed NICU caregivers in a convenience sample of 12 hospitals from a single health care system, using the Safety Attitudes Questionnaire (SAQ). The six scales of the SAQ include teamwork climate, safety climate, job satisfaction, stress recognition, perception of management, and working conditions. For each NICU we calculated scale means, standard deviations and percent positives (percent agreement). results We found substantial variation in safety culture domains among participating NICUs. A composite mean score across the six safety culture domains ranged from 56.3 to 77.8 on a 100-point scale and NICUs in the top four NICUs were significantly different from the bottom four (p < .001). Across the six domains, respondent assessments varied widely, but were least positive on perceptions of management (3–80% positive; mean 33.3%) and stress recognition (18–61% positive; mean 41.3%). Comparisons of SAQ scale scores between NICUs and a previously published cohort of adult ICUs generally revealed higher scores for NICUs. Physicians composite scores were 8.2 (p = .04) and 9.5 (p =.02) points higher than nurses and ancillary personnel. conclusion Significant variation and scope for improvement in safety culture exists among this sample of NICUs. The NICU variation was similar to variation in adult ICUs, but NICU scores were generally higher than adult ICU scores. Future studies should validate whether safety culture as measured with the SAQ correlates with clinical and operational outcomes in the NICU setting.