Residual biospecimens that are stored in hospitals' diagnostic specimen archives can be used for scientific research under strict legal and ethical regulations. In the Netherlands, a Code of Conduct governs responsible secondary use of residual biospecimens. However, implementation of this Code seems to be challenging. This study aims to explore the most important factors that facilitate or hinder the implementation of the Code. In addition, it investigates what is needed to further foster the responsible use of residual biospecimens. A mixed-methods design was used. Questionnaires were sent out to pathologists, patient information centers, physicians, researchers, data protection officers (DPOs), members of research ethics committees, and members of the boards of directors of all hospitals in the Netherlands (81 hospitals). To further investigate the barriers and facilitators, interviews were conducted with pathologists, patient information centers, physician-researchers, DPOs, review boards, research coordinators, and quality managers of pathology departments. In total, 246 respondents filled out the questionnaire and 36 interviews were conducted. Major barriers for implementing were a lack of resources (time, money), a lack of attention for responsible use, and a lack of practical knowledge (knowing what to do, where to go with questions). In contrast, the perception that implementing the Code was necessary, either by the respondent or by colleagues, was considered "a driver" for implementation. Practical instruments such as checklists and roadmaps were considered necessary to foster implementation; however, the creation of such instruments was hindered by a lack of clear-cut answers regarding legal aspects. Therefore, more clarity and harmonization on how to interpret both the Code and legislation regarding secondary use were considered necessary.