Calcineurin is a calcium-regulated serine-threonine protein phosphatase that controls developmental and inducible biological responses in diverse cell types, in part through activation of the transcription factor nuclear factor of activated T cells (NFAT). In skeletal muscle, calcineurin has been implicated in the regulation of myoblast differentiation, hypertrophy of mature myofibers, and fiber type switching in response to alterations in intracellular calcium concentration. However, considerable disagreement persists about the functional role of calcineurin signaling in each of these processes. Here we evaluated the molecular phenotypes of skeletal muscle from both calcineurin A␣ and calcineurin A gene-targeted mice. Calcineurin A␣ was observed to be the predominant catalytic isoform expressed in nearly all skeletal muscles examined. Neither calcineurin A␣ or A null mice showed any gross growth-related alterations in skeletal muscle, nor was fiber size or number altered in glycolytic/fast muscle types. In contrast, both calcineurin A␣ and A gene-targeted mice demonstrated an alteration in myofiber number in the soleus, an oxidative/slow-type muscle. More significantly, calcineurin A␣ and A gene-targeted mice showed a dramatic down-regulation in the oxidative/slow fiber type program in multiple muscles (both slow and fast). Associated with this observation, NFAT-luciferase reporter transgenic mice showed significantly greater activity in slow fiber-containing muscles than in fast. However, only calcineurin A␣ null mice showed a defect in NFAT nuclear occupancy or NFAT-luciferase transgene activity in vivo. Collectively, our results suggest that calcineurin signaling plays a critical role in regulating skeletal muscle fiber type switching but not hypertrophy. Our results also suggest that fiber type switching occurs through an NFAT-independent mechanism.Calcineurin is a calcium-calmodulin-activated serine-threonine phosphatase composed of a catalytic A subunit (59 to 62 kDa) and a calcium-binding regulatory B subunit (19 kDa). Three catalytic genes (A subunit) have been identified in vertebrate species, of which calcineurin A␣ and calcineurin A are ubiquitously expressed, while calcineurin A␥ expression is restricted to the testis and brain (11,23,38). Receptor stimulation that promotes a sustained elevation in intracellular calcium concentration leads to a direct activation of calcineurin, which in turn facilitates alterations in gene expression through transcriptional effector proteins (11,23,38). One such mechanism involves a family of transcriptional regulators referred to as nuclear factor of activated T cells (NFAT), which are normally sequestered in the cytoplasm in a hyperphosphorylated state (36). Activated calcineurin then directly binds NFAT transcription factors in the cytoplasm, resulting in their dephosphorylation and subsequent translocation into the nucleus. Once in the nucleus, NFAT factors function as important coinducers of calcium-activated, inducible gene expression in multiple cell typ...