. NFAT activation by membrane potential follows a calcium pathway distinct from other activity-related transcription factors in skeletal muscle cells. Am J Physiol Cell Physiol 294: C715-C725, 2008. First published January 9, 2008 doi:10.1152/ajpcell.00195.2007.-Depolarization of skeletal muscle cells triggers intracellular Ca 2ϩ signals mediated by ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors. Previously, we have reported that K ϩ -induced depolarization activates transcriptional regulators ERK, cAMP response element-binding protein, c-fos, c-jun, and egr-1 through IP3-dependent Ca 2ϩ release, whereas NF-B activation is elicited by both ryanodine and IP3 receptor-mediated Ca 2ϩ signals. We have further shown that field stimulation with electrical pulses results in an NF-B activation increase dependent of the amount of pulses and independent of their frequency. In this work, we report the results obtained for nuclear factor of activated T cells (NFAT)-mediated transcription and translocation generated by both K ϩ and electrical stimulation protocols in primary skeletal muscle cells and C2C12 cells. The Ca 2ϩ source for NFAT activation is through release by ryanodine receptors and extracellular Ca 2ϩ entry. We found this activation to be independent of the number of pulses within a physiological range of stimulus frequency and enhanced by long-lasting low-frequency stimulation. Therefore, activation of the NFAT signaling pathway differs from that of NF-B and other transcription factors. Calcineurin enzyme activity correlated well with the relative activation of NFAT translocation and transcription using different stimulation protocols. Furthermore, both K ϩ -induced depolarization and electrical stimulation increased mRNA levels of the type 1 IP3 receptor mediated by calcineurin activity, which suggests that depolarization may regulate IP3 receptor transcription. These results confirm the presence of at least two independent pathways for excitationtranscription coupling in skeletal muscle cells, both dependent on Ca 2ϩ release and triggered by the same voltage sensor but activating different intracellular release channels. nuclear factor of activated T cells transcription; nuclear factor of activated T cells translocation; calcineurin; inositol 1,4,5-trisphosphate receptor; ryanodine receptor IN SKELETAL MUSCLE CELLS, depolarization by high K ϩ induces Ca 2ϩ release from intracellular stores mediated both by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 Rs) (11,18). The kinetics of the resulting Ca 2ϩ