BACKGROUND: The use of telemedicine in acute stroke care can facilitate rapid access to treatment, but the work required to embed any new technology into routine practice is often hidden, and can be challenging. We aimed to collate recommendations and resources to support telestroke implementation. METHODS: Systematic search of healthcare databases and the Internet to identify descriptions of the implementation of telestroke projects; interviews with key stakeholders during the development of one UK telestroke network. Supporting documentation from existing projects was analysed to construct a framework of implementation stages and tasks, and a toolkit of documents. Interviews and literature were analysed with other data sources using Normalisation Process Theory as described in the e-Health Implementation Toolkit. RESULTS: 61 telestroke projects were identified and contacted. Twenty projects provided documents, 13 with published research detailing four stages of telestroke system development, implementation, use, and evaluation. Interviewees identified four main challenges: engaging and maintaining the commitment of a wide range of stakeholders across multiple organisations; addressing clinicians perceptions of evidence, workload, and payback; managing clinical and technical workability across diverse settings; and monitoring how the system is used and reconfigured by users. CONCLUSIONS: Information to guide telestroke implementation is sparse, but available. By using multiple sources of data, sufficient information was collated to construct a web-based toolkit detailing implementation tasks, resources and challenges in the development of a telestroke system for assessment and thrombolysis delivery in acute care. The toolkit is freely available online.