The class II histone deacetylases, HDAC4 and HDAC5, directly bind to and repress myogenic transcription factors of the myocyte enhancer factor-2 (MEF-2) family thereby inhibiting skeletal myogenesis. During muscle differentiation, repression of gene transcription by MEF-2/HDAC complexes is relieved due to calcium/calmodulin-dependent (CaM) kinase-induced translocation of HDAC4 and HDAC5 to the cytoplasm. MEF-2 proteins and HDACs are also highly expressed in the nervous system and have been implicated in neuronal survival and differentiation. Here we investigated the possibility that the subcellular localization of HDACs, and thus their ability to repress target genes, is controlled by synaptic activity in neurones. We found that, in cultured hippocampal neurones, the localization of HDAC4 and HDAC5 is dynamic and signal-regulated. Spontaneous electrical activity was sufficient for nuclear export of HDAC4 but not of HDAC5. HDAC5 translocation to the cytoplasm was induced following stimulation of calcium flux through synaptic NMDA receptors or L-type calcium channels; glutamate bath application (stimulating synaptic and extrasynaptic NMDA receptors) antagonized nuclear export. Activity-induced nucleocytoplasmic shuttling of both HDACs was partially blocked by the CaM kinase inhibitor KN-62 with HDAC5 nuclear export being more sensitive to CaM kinase inhibition than that of HDAC4. Thus, the subcellular localization of HDACs in neurones is specified by neuronal activity; differences in the activation thresholds for HDAC4 and HDAC5 nuclear export provides a mechanism for input-specific gene expression.