Research capacity building (RCB) in Australia has recently focussed on strategies that take a whole of system approach to developing research culture at individual, team and organisation levels. Although a theoretical framework exists, no tool has been published that quantitatively measures the effectiveness of RCB interventions aimed at these three levels. A sample of 134 allied health workers was used to validate the research capacity and culture (RCC) tool. Item level analysis was undertaken using Cronbach's α and exploratory factor analysis, and test-retest reliability was examined using intra-class correlations (ICC). The tool had one factor emerge for each domain, with excellent internal consistency for organisation, team and individual domains (α=0.95, 0.96 and 0.96 respectively; and factor loadings ranges of 0.58-0.89, 0.65-0.89 and 0.59-0.93 respectively). The overall mean score (total) for each domain was: 5.4 (inter-quartile range 3.9-7.7), 4.4 (IQR 2.6-6.1) and 3.9 (IQR 2.9-6) for the organisation, team and individual domains respectively. Test-retest reliability was strong for each domain: organisation ICC=0.77, team ICC=0.83 and individual ICC=0.82. The RCC tool has three domains measuring research capacity and culture at organisation, team and individual levels. It demonstrates excellent internal consistency and strong test-retest reliability.