BackgroundThe organizational culture (shared beliefs, perceptions, and values) of teams informs their behaviours and practices. Little is known about organizational culture for resuscitation teams. Our objective was to develop a reliable and valid resuscitation-specific organizational culture instrument (ROCI) with the goal of improving team performance. MethodsUsing Neonatal Resuscitation Program principles, literature review, and discussion of existing culture measures with experts, we identified organizational culture components for resuscitation and adapted existing measures to resuscitation. We developed a ROCI with five subscales (role clarity, shared-mental models, closed-loop communication, team adaptability, and psychological safety) and administered it to neonatal resuscitation team members across a hospital network. Survey psychometric assessment included reliability analyses (Cronbach’s α, Pearson correlation coefficients) and validity testing (confirmatory factor analysis [CFA] and regression models examining the association of culture with implementation outcomes: climate and perceived success). ResultsAcross 11 hospitals there were 318 complete responses (41 % response rate). Of the 22-items tested, 18 were retained after iterative psychometric assessment. The ROCI had excellent overall reliability (Cronbach’s α = 0.994) and very good subscale reliability (Cronbach’s α = 0.789–0.867). The CFA goodness-of-fit statistics confirmed five constructs (subscales). At the individual-level, the ROCI and all subscales were associated with both implementation outcomes. At the hospital-level, the ROCI overall and three subscales were associated with perceived success. ConclusionThe ROCI is a reliable and valid measure of the organizational culture of resuscitation teams. Future ROCI assessments may provide a foundation to inform culture change initiatives to improve resuscitation quality and outcomes across populations and contexts.