Objectives: The main aim of the study was to explore the potential sources of variation and understand the meaning of safety climate for nursing practice in acute hospital settings in the UK. Design: A sequential mixed methods design included a cross-sectional survey using the Safety Climate Questionnaire (SCQ) and thematic analysis of focus group discussions. Confirmatory Factor Analysis (CFA) was used to validate the factor structure of the SCQ. Factor scores were compared between nurses working in operating theatres, critical care and ward areas. Results from the survey and the thematic analysis were then compared and synthesised. Setting: A London University. Participants: 319 registered nurses working in acute hospital settings completed the SCQ and a further 23 nurses participated in focus groups. Results: CFA indicated that there was a good model fit on some criteria (χ2 = 1683.699, df 824, p < 0.001; χ2/ df = 2.04; RMSEA = 0.058) but a less acceptable fit on Comparative Fit Index (CFI) = 0.804. There was a statistically significant difference between clinical specialisms in Management Commitment (F [4,266] = 4.66, p = 0.001). Nurses working in operating theatres had lower scores compared with ward areas and they also reported negative perceptions about management in their focus group. There was significant variation in scores for Communication across clinical specialism (F [4,266] = 2.62, p = 0.035) but none of the pair-wise comparisons achieved statistical significance. Thematic analysis identified themes of Human Factors, Clinical Management and Protecting Patients. The System and the Human Side of Caring was identified as a meta-theme. Conclusions: The results suggest that the SCQ has some utility but requires further exploration. The findings indicate that safety in nursing practice is a complex interaction between safety systems and the social and interpersonal aspects of clinical practice.