The 'triple helix' of the university-industry-government relationship and habitat are accepted as important determinants of innovation and entrepreneurship. However, empirical explorations of the effects of these variables and their interrelationships on regional entrepreneurial activities are highly limited. To fill this gap, we investigate the effect of the triple helix system and habitat on birth and death rates of U.S. firms at the state level. As expected, we find that industrial R&D expenditure plays an important role in promoting regional firm birth. However, university and government R&D also generate a synergistic effect that indirectly influences regional firm birth rates. In addition, we find that the synergy between university and industrial R&D enhances the sustainability of firms, while the interactions between (1) university and government R&D and (2) government and industrial R&D are associated with an increase in firm death. Other factors linked to more favorable conditions for firm formation include higher educational attainment in a region, lower tax rate, and habitat factors affecting quality of life, such as lower housing prices and higher rates of health insurance coverage. In regions with high entrepreneurial activity, we find positive synergistic effects of the interactions between (1) university and government R&D and (2) university and industrial R&D on firm birth rate, suggesting that university R&D plays an important role as an 'entrepreneurial mediator' among the three spheres in the triple helix system. In low entrepreneurial regions, the only triple helix system factors significantly influencing firm birth rate were tax rate. This finding suggests that the independent and interdependent components of the triple helix system and habitat are less powerful in low entrepreneurial regions. (C) 2011 Elsevier B.V. All rights reserved.