<span class="paragraphSection"><div class="boxTitle">Abstract</div>Antisense oligonucleotides (ASOs) are an important therapeutic modality for neurological diseases. Some ASOs cause transient neurobehavioral responses acutely following intrathecal delivery to the central nervous system (CNS). We characterized a subset of these responses, including hypoactivity, spinal reflex loss, paresis, sedation, and ataxia, that are suggestive of neuronal inhibition in rodents and non-human primates. Across species, inhibition-like responses peaked ∼3 h post-ASO delivery, reversed within 24 h with no sequelae, and could be quantified using simple neurobehavioral scales. Acute inhibition was dose-responsive and was abrogated with lower phosphorothioate and guanine content in ASOs. Acutely inhibitory ASOs transiently disrupted motor pathway neurotransmission <span style="font-style: italic;">in vivo</span> and suppressed firing in primary neural cultures. <span style="font-style: italic;">In vitro</span> firing rate suppression of &gt;60% predicted high <span style="font-style: italic;">in vivo</span> acute inhibition scores and was reversed immediately with addition of varying excitatory agents or upon ASO washout. Peak acute inhibition <span style="font-style: italic;">in vivo</span> coincided with peak CNS tissue concentrations of ASO and abated as ASO was internalized by parenchymal cells and cleared from the extracellular space. We propose transient high extracellular concentrations block synaptic transmission via non-specific protein binding of phosphorothioate ASOs. Our results define a comprehensive framework for quantifying and mitigating ASO-mediated acute inhibition.</span>