Acetylation is an important post-translational modification (PTM). Lysine acetylation is a reversible PTM, where deacetylation is catalyzed by histone deacetylases (HDACs). Histone deacetylase function is crucial for a correctly functioning cell as aberrant acetylation, or deacetylation, has been linked to cancer, diabetes, neurodegeneration, and auto-immune disorders. Yet information about proper regulation of these enzymes is limited. Regulation of HDAC activity and selectivity has been proposed to include: the identity of the divalent active site metal ion, post-translational modifications, and protein interactions to form stable multi-protein complexes. HDAC activity and selectivity is further influenced by substrate amino acid sequence. This thesis explores how these different regulatory measures impact HDAC activity and selectivity. The biochemically well-characterized HDAC8 was used to investigate novel HDAC inhibitors and it was found that the identity of the active site divalent metal ion plays an important role in determining inhibitor selectivity. The identification and characterization of inhibitors with selective metal-binding groups, particularly Fe(II)-HDAC8 selective inhibitors, demonstrates structural differences between different HDAC8 metalloforms. This work also identified that the tropolone metal binding group potently inhibits HDAC8. To examine the impact of post-translational modifications and protein interactions on deacetylase activity and selectivity, a simplified CoREST complex including HDAC1 was reconstituted. In vitro HDAC1 complex formation significantly increases deacetylase activity (>10-fold) in comparison to HDAC1 in isolation. The presence of post-translational modifications, specifically phosphorylation, was found to impact substrate selectivity with the identification of a phosphorylation-specific acetylation site, without preventing complex formation. Finally, to explore the sequence-level substrate selectivity of HDAC6, we successfully constructed a structure-based model of the catalytic domain of HDAC6. This model was used to predict novel substrates that were then validated using peptide mimics. These data demonstrated that the substrate selectivity of HDAC6 is more promiscuous than HDAC8. The comparison of the activity of the single catalytic domain of HDAC6 with HDAC6 containing both catalytic domains demonstrates that the different structural components influence the activity and substrate selectivity profile of the enzyme. The findings discussed within this thesis illustrate several regulatory factors impart a sizeable contribution to deacetylase activity and selectivity. Such factors include structural components, including cofactors and post-translational modifications, in addition to protein interactions. The contribution of this thesis to the growing knowledge of how HDACs are regulated provides insight into the enzymes’ biological function to lead to the development of more effective therapeutic interventions.