Nuclear factor of activated T cells (NFAT) is a family of transcription factors, named NFATc1-4, that is activated in response to calcium signals. The NFAT family members are sequentially similar but are found to play different roles in the skeletal muscle, where they regulate phenotype and size of the muscle fibers. In the present study, the aim was to explore and characterize the members of the NFAT family in skeletal muscle. The transcriptional potential of the family members was assessed in vitro by transfecting HEK293 cells with a stably integrated 5x Gal-recognition elements followed by the gene coding for Firefly-Luciferase. In addition, the influence of the interaction partner and coregulator p300 on NFAT was assessed in the same system. Furthermore, the NFAT activity and the expression of NFAT and selected NFAT target genes in skeletal muscle were explored in response to voluntary endurance training in vivo with NFAT luciferase reporter mice and NMRI, respectively. The results show that all NFAT family members have a transcriptional activating potential; however, NFATc1 is a significantly less potent activator and is also the only NFAT that display a repressive potential in vitro. Subsequently, cotransfection of p300 together with NFAT increases the transactivational potential of all NFATs in the HEK293 system. The in vivo analysis found that the NFAT activity is increased in soleus and plantaris in response to voluntary exercise, while expression of the NFATs are decreased in plantaris in response to exercise, pointing towards an interesting regulatory mechanism to control the NFAT level in the muscle cell.