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Background Building research capacity within health care services is crucial to generate a solid evidence base for decision-making in policy and practice. For hospital and healthcare services to foster a strong research culture, a multi-faceted approach to research capacity building needs to be supported at organisational, team and individual levels. Aim To understand differences in the research culture and capacity between professional streams within a large tertiary public health service. Methods A cross-sectional observational study was conducted within a large metropolitan tertiary hospital, using the Research Culture and Capacity (RCC) tool to identify organisational, team, and individual level factors. All employees, including medical, nursing, allied health, corporate, administration and professional staff were included. Results In total, 739 health service employees completed the survey, including 218 Allied Health practitioners, 99 medical doctors, 312 nurses, and 107 corporate, administrative and professional employees. Differences were observed relative to professional stream with Allied Health and Nursing rating organisational factors highest, whilst Medical rated individual factors highest, and Professional/corporate employees rated organisational, team and individual levels equally. Overall analysis, inclusive of all streams, revealed significant differences between organisational and team (X 2 =5.100, p<.001) and organisational and individual (X 2 =3.301, p<.001) level factors, with organisational level factors being significantly higher. Conclusion Research culture and capability varies across allied health, medical, and nursing streams. A focus on addressing barriers and enablers through targeted activities tailored to profession, particularly at the team level, may improve research activity and outcomes.