Sustainability has become one the main drivers of innovation and many regions in the world aim to transform into a ‘sustainable innovation region’. Culture is an essential element of the innovation environment in a sustainable innovation policy. This article discusses recent insights in the theoretical and empirical foundation of innovation policies aimed at developing ‘cultures of sustainable innovation’. A multidisciplinary approach based on the dilemma paradigm of enquiry is used to develop a dynamic framework on how to foster sustainable innovation. The approach is applied in an assessment of the sustainable innovation culture in two regions: Silicon Valley and Southeast Netherlands. It is concluded that Regional Innovation Systems can be assessed by evaluating to what extent a dynamic balance is established on each of the innovation culture dilemmas. However, copying ‘success formulas’ for sustainable innovation from other regions is impossible. The dynamic balance depends on history and culture of the region and the continuous interaction with the external environment.