Adult skeletal muscle fibers can be divided into fast and slow twitch subtypes on the basis of specific contractile and metabolic properties, and on distinctive patterns of muscle gene expression. The calcium, calmodulin-dependent protein phosphatase, calcineurin, stimulates slow fiber-specific genes (myoglobin (Mb), troponin I slow) in cultured skeletal muscle cells, as well as in transgenic mice, through the co-operation of peroxisome-proliferation-activator receptor gamma co-activator 1alpha (PGC1alpha) myocyte enhancer factor 2 (MEF2), and nuclear factor of activated T cells (NFAT) transcription factors. Specific protein kinase C isoforms have been shown to functionally co-operate with calcineurin in different cellular models. We investigated whether specific protein kinase C isoforms are involved in calcineurin-induced slow skeletal muscle gene expression. By pharmacological inhibition or exogenous expression of mutant forms, we show that protein kinase C theta (the protein kinase C isoform predominantly expressed in skeletal muscle) is required and co-operates with calcineurin in the activation of the Mb promoter, as well as in the induction of slow isoforms of myosin and troponin I expression, in cultured muscle cells. This co-operation acts primarily regulating MEF2 activity, as shown by using reporter gene expression driven by the Mb promoter mutated in the specific binding sites. MEF2 activity on the Mb promoter is known to be dependent on both PGC1alpha and inactivation of histone deacetylases (HDACs) activity. We show in this study that protein kinase C theta is required for, even though it does not co-operate in, PGC1alpha-dependent Mb activation. Importantly, protein kinase C theta regulates the HDAC5 nucleus/cytoplasm location. We conclude that protein kinase C theta ensures maximal activation of MEF2, by regulating both MEF2 transcriptional complex formation and HDACs nuclear export.