The protein kinase PKD1 has recently been linked to slow fibre-type gene expression in fast skeletal muscle through phosphorylation of class II histone deacetylase (HDAC) molecules, resulting in nuclear efflux of HDAC and consequent activation of the transcription factor MEF2. However, possible upstream activators of PKD, and the time course and signalling pathway of downstream effectors have not been determined in skeletal muscle. Using fluorescent fusion proteins HDAC5-green fluorescent protein (GFP) and PKD1-mPlum expressed in fibres isolated from predominantly slow soleus muscle and maintained for 4 days in culture, we now show that alpha-adrenergic receptor activation by phenylephrine causes a transient, PKD-dependent HDAC5-GFP nuclear efflux. Concurrent to this response, PKD1-mPlum transiently redistributes from cytoplasm to plasma membrane and nuclei, and back, during 2 h exposure to phenylephrine. The recovery may reflect alpha-receptor desensitization. In contrast, the phorbol ester PMA (phorbol-12-myristate-13-acetate, a pharmacological mimic of the downstream mediator diacylglycerol in alpha-adrenergic signalling), caused continuous PKD-dependent HDAC5-GFP nuclear efflux and maintained PKD1-mPlum redistribution. In the absence of expressed HDAC, PMA increased histone H3 acetylation and increased MEF2 reporter activity in a PKD-dependent manner, consistent with PKD phosphorylation of endogenous HDAC(s) and reduced nuclear HDAC activity due to HDAC nuclear efflux. HDAC5-GFP did not respond to PMA in fibres from predominantly fast flexor digitorum brevis (FDB) muscle, but did in FDB fibres expressing exogenous PKD1. Our results demonstrate that a PKD-mediated signalling pathway for HDAC nuclear efflux is activated in slow skeletal muscle through adrenergic input, which is typically active in parallel with motor neurone input during muscular activity.