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Purpose Entrepreneurial and strategic actions are crucial for wealth creation, and the business opportunity is a critical factor in this process. The purpose of this paper is to explore the role of the firm’s strategic posture in the relationship between individual alertness and opportunity identification within an existing firm. This approach contributes to entrepreneurship theory building through a multilevel study. Design/methodology/approach The quantitative research focuses on understanding the mediating role of an organization’s strategic posture in the opportunity identification process. Using a sample of 276 firms, this study tests a two-level model to explain opportunity identification. Findings The findings provide empirical evidence that a firm’s strategic posture mediates the relationship between individual alertness and opportunity identification. Furthermore, this study finds differences in the mediating role of a firm’s strategic posture through which entrepreneurs and managers affect opportunity identification. Years after the creation of startup, the entrepreneurs still exhibit entrepreneurial characteristics that affect opportunity identification. The findings provide evidence that entrepreneurs foster an internal culture and set of values that are more favorable to radical innovation, compared to managers who favor incremental and less risky projects. Practical implications The findings suggest the possibility for new theory building that can improve the fields of entrepreneurship and management research. Moreover, the proposed model constitutes a new approach to analyze the mediating role of an organization’s strategic posture in the opportunity identification process. Originality/value This paper provides an original approach to literature in exploring the relationship between entrepreneurial alertness and firm’s strategic posture in explaining the opportunity identification process. This work will help expand the theory building that explores differences between managers and entrepreneurs in organizations.