To test for a role of the calcineurin-NFAT (nuclear factor of activated T cells) pathway in the regulation of fiber type-specific gene expression, slow and fast muscle-specific promoters were examined in C2C12 myotubes and in slow and fast muscle in the presence of calcineurin or NFAT2 expression plasmids. Overexpression of active calcineurin in myotubes induced both fast and slow muscle-specific promoters but not non-muscle-specific reporters. Overexpression of NFAT2 in myotubes did not activate muscle-specific promoters, although it strongly activated an NFAT reporter. Thus overexpression of active calcineurin activates transcription of muscle-specific promoters in vitro but likely not via the NFAT2 transcription factor. Slow myosin light chain 2 (MLC2) and fast sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA1) reporter genes injected into rat soleus (slow) and extensor digitorum longus (EDL) (fast) muscles were not activated by coinjection of activated calcineurin or NFAT2 expression plasmids. However, an NFAT reporter was strongly activated by overexpression of NFAT2 in both muscle types. Calcineurin and NFAT protein expression and binding activity to NFAT oligonucleotides were different in slow vs. fast muscle. Taken together, these results indicate that neither calcineurin nor NFAT appear to have dominant roles in the induction and/or maintenance of slow or fast fiber type in adult skeletal muscle. Furthermore, different pathways may be involved in muscle-specific gene expression in vitro vs. in vivo.