Introduction: Breast cancer (BC) is the most common cancer in the world and the deadliest among women in France (WHO 2020). It can be classified into molecular subtypes based on the expression of three receptors: epidermal growth factor type 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER). 70% of cases express ER, linked to a 94% 5-year survival. However, when the BC relapses, the survival rate drops to 23%, with 36% showing ESR1 mutations, mainly in the ligand-binding domain (LBD), inducing endocrine resistance. Methods: One way to investigate these mutations is positron emission tomography (PET) using the 18fluoroestradiol ([¹⁸F]-FES) radiotracer, capable of binding to the ER's LBD. However, its ability to bind to ER mutants remains unclear. The purpose of this study is to examine the affinity of [¹⁸F]-FES for ER mutants to determine its potential for diagnostic use and therapeutic monitoring of patients. For this, six clones expressing the most frequently found mutants in metastatic stage patients were generated (Y537S/C/N, D538G, E380Q, and L536H). Characterization studies of the inducibility of this Tet-ON model and its functionality were conducted in vitro and in vivo. Finally, the binding of the [¹⁸F]-FES radiotracer to the wild-type ER expressed by our model was investigated. Results/Discussion: All in vitro analyses confirmed the inducibility of our model under doxycycline stimulation (1000 ng/ml for 24h). In vivo inducibility was also validated through orthotopic injection in SCID mice. Furthermore, we demonstrated that the expressed ER protein is fully functional, and the Y537, D538, and L536 mutants are constitutively active, as described in the literature (Albert Grinshpun et al. 2023). Micro PET confirmed [¹⁸F]-FES binding to induced ER after orthotopic injection. The study of the Standardized Uptake Value (SUV) of [¹⁸F]-FES on ER mutants is currently under analysis by micro PET.  Affinity studies using microscale thermophoresis (MST) to assess ER mutants' interaction with estradiol are also in progress. Conclusion: All these data should permit us to specify the binding capacity of [¹⁸F]-FES to different mutated ER receptors conferring endocrine resistance. This would strengthen the interest in FES examination, showing not only if cells no longer express ER but also if those that remain ER+ have mutated and/or if the proposed treatment promotes the occurrence of these mutations.