Structurally distinctive within the Zn 2+-dependent HDAC family, HDAC6 and HDAC10 feature two deacetylase domains. In HDAC6, both domains are active with different functions: deacetylase domain 1 (DD1) focuses on deacetylating substrates with acetylated lysine at their C terminus, 1 while deacetylase domain 2 (DD2) exclusively targets peptides featuring an internal acetylated lysine residue. Conversely, HDAC10 possesses an active polyamine deacetylase (PDAC) domain and an inactive pseudodeacetylase (ΨDAC) domain. Key residues in the active site of HDAC10, such as E274 (zebrafish numbering), contribute to its specificity for polyamine substrates. The unique ηA2 helix and a specific loop induce steric constriction in the active site, thereby influencing its deacetylase activities. Despite differences in substrate selectivity, crystal structures reveal a typical assembly pattern for HDAC6 and HDAC10, opening avenues for developing molecular tools to achieve dual inactivation for both targets.