Raising cytosolic Ca2+ induces an increase in mitochondrial biogenesis in myotubes. This phenomenon mimics the adaptive responses of skeletal muscle to exercise. It has been hypothesized that increases in cytosolic Ca2+ during motor nerve activity stimulate mitochondrial biogenesis by activating calcineurin. Overexpression of constitutively active calcineurin increases expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and induction of genes involved in mitochondrial energy metabolism in muscle cells. The purpose of this study was to determine whether calcineurin plays a role in the stimulation of mitochondrial biogenesis by exercise. Rats were exercised on 5 successive days by means of swimming. Inhibition of calcineurin with cyclosporin did not prevent the exercise-induced increases in PGC-1alpha and a range of mitochondrial proteins. In contrast to the other mitochondrial proteins, the increases in cytochrome oxidase (COX)-I and -IV proteins were blocked by cyclosporin treatment. This inhibitory effect of cyclosporin occurred at the posttranscriptional level, as evidenced by normal increases in COX-I and COX-IV mRNAs in response to exercise in the cyclosporin-treated rats. This toxic effect of cyclosporin may account for the decrease in muscle respiratory capacity reported to occur with cyclosporin treatment. In conclusion, inhibition of calcineurin does not prevent the exercise-induced increase in mitochondrial biogenesis in skeletal muscles, providing evidence that the adaptive response is not mediated by activation of calcineurin.