Site-directed spin labeling (SDSL) EPR spectroscopy has emerged as a powerful technique to study the structure and conformational dynamics of biomolecules in vitro; however, the application of this technique in live cells still remains challenging. The goal of this proposal is the development and application of SDSL EPR spectroscopy in vivo, which allows addressing problems of cell biology not accessible by in vitro experiments. Enzymatic spin labeling techniques and click chemistry will be developed to bind spin labels to selected sites of proteins in live cells. Furthermore, new spin labels as alternatives to the reduction-sensitive nitroxide systems will be systematically evaluated for their applicability in SDSL EPR in vivo. As a model system, the interaction and conformational changes of the bacterial toxin colicin A and the corresponding immunity protein Cai will be studied in live Escherichia coli cells. Further methodological developments comprise the application of microresonators for the enhancement of the EPR detection sensitivity in “in cell” experiments.