Transcription factor nuclear factor of activated T cells NFATc (NFATc1, NFAT2) may contribute to slow-twitch skeletal muscle fiber type-specific gene expression. Green fluorescence protein (GFP) or FLAG fusion proteins of either wild-type or constitutively active mutant NFATc [NFATc(S-->A)] were expressed in cultured adult mouse skeletal muscle fibers from flexor digitorum brevis (predominantly fast-twitch). Unstimulated fibers expressing NFATc(S-->A) exhibited a distinct intranuclear pattern of NFATc foci. In unstimulated fibers expressing NFATc-GFP, fluorescence was localized at the sarcomeric z-lines and absent from nuclei. Electrical stimulation using activity patterns typical of slow-twitch muscle, either continuously at 10 Hz or in 5-s trains at 10 Hz every 50 s, caused cyclosporin A-sensitive appearance of fluorescent foci of NFATc-GFP in all nuclei. Fluorescence of nuclear foci increased during the first hour of stimulation and then remained constant during a second hour of stimulation. Kinase inhibitors and ionomycin caused appearance of nuclear foci of NFATc-GFP without electrical stimulation. Nuclear translocation of NFATc-GFP did not occur with either continuous 1 Hz stimulation or with the fast-twitch fiber activity pattern of 0.1-s trains at 50 Hz every 50 s. The stimulation pattern-dependent nuclear translocation of NFATc demonstrated here could thus contribute to fast-twitch to slow-twitch fiber type transformation.