The intentional use of organophosphate (OP) nerve agents and pesticides pose a significant health threat through their inhibition of human acetylcholinesterase (hAChE), which serves a critical role in neurotransmission. Their recent use in the assassination of the half-brother of the North Korean leader, the ongoing Syrian Civil War, and the United Kingdom has reaffirmed their relevant toxicity. Inhibition of AChE occurs when the catalytic serine becomes covalently modified by the nerve agent adduct. AChE displays stereoselectivity for OP nerve agent binding resulting in stereospecific toxicities of these agents. Oxime reactivators are one of the current treatments for OP exposure, which seek to reverse nerve agent conjugation of the hAChE active site serine prior to aging. However, the therapeutic efficacy of these agents varies based on the OP used, the stereochemistry of the OP, and the origin species of AChE. Currently the factors that influence reactivator efficacy are not well-understood including the influence of AChE's stereoselectivity on the accommodation of OP nerve agent stereoisomers and their impact on subsequent reactivation. Due to these issues, there is a strong need to develop novel reactivators. However, until now there were lingering questions about the mechanism of reactivation. In addition, the means to accommodate certain nerve agents and reactivators into the active site is potentially due to the flexibility of certain regions of hAChE, but these dynamics are not well-understood. This study aims to fill these major gaps in knowledge to provide insight into the dynamics of the active site and its ability to accommodate a wide variety of OP nerve agents and reactivators. Through modified Ellman's assays and X-ray crystallography, this study examines the impact of hAChE's stereoselectivity on inhibition and reactivation, the influence of different nerve agents and their subsequent reactivation on hAChE's active site, and the structural and biochemical effects of monomerizing hAChE via site-directed mutagenesis. These studies provided the first structural insight into the ability of hAChE's active site to accommodate nerve agents and reactivators, elucidated some of the factors that influence this accommodation, and developed a tool for assessing the flexibility and dynamics of the active site.