Distinct depolarization patterns differentially regulate the slow- and fast-twitch properties of skeletal muscle fibers by regulating intracellular Ca²⁺ levels. The transcription factor NFAT and NF-B are known to be calcium-regulated factors that differentially regulate downstream target genes in T-cells. Here we show that NFAT and NF-B may also differentially regulate fast muscle genes in response to patterned electrical activity. To map the activity responsive elements in the Troponin I (TnI) gene, we have generated a series of deletions and mutations in the TnI fast intronic regulatory element (FIRE) and measured transcription rates. Extracellular electrodes were implanted in solei muscles of rats to stimulate muscles transfected with the different TnI FIRE mutations. Transcription of the GFP reporter in muscles stimulated with matching (slow) or mismatching (fast) patterns of electrical activity was measured in live myofibers to map the responsive elements. Our analysis revealed that FIRE transcription rate increased when stimulated with a fast activity pattern, while slow activity pattern showed no change. Mutation in a NFAT like site in FIRE resulted in increased activity in response to slow activity, whereas increased transcription rate due to fast activity was lost when the mutation resided in a NF-B like site. EMSA showed that NFAT and NF-B bound to their putative sites in FIRE. siRNA knockdown of NFAT resulted in increased FIRE activity in soleus. Knockdown of NF-B resulted in decreased activity in EDL. These results suggest that NFAT is responsible for down-regulation of fast-specific Troponin I gene transcription in response to slow twitch muscle activity, whereas NF-B is necessary for the up-regulation in response to fast twitch muscle activity.