Abstract Cholinergic contribution to pupil-linked arousal Behavioral state, including attention and arousal, exerts heavy influences on neural representation, perception, cognition, and behavioral performance and is regulated by several neuromodulatory systems, including the cholinergic systems and the locus coeruleus-norepinephrine (LC-NE) system. Abnormal activity in the cholinergic systems has been implicated in major clinical disorders that affect millions of people, including schizophrenia, Parkinson’s disease (PD), and depression. Non-luminance mediated changes in pupil size have been known to co-vary with mental processing underlying behavior for decades. Recent work highlighted that pupil dynamics were able to track rapid fluctuation of cortical arousal state. Therefore, change in pupil size under constant illumination has been widely used as a non-invasive readout of the activation of certain central arousal circuits related to pupil size, and thus is used to index pupil-linked arousal. Atypical pupil dynamics have been reported in the aforementioned neurological disorders. However, the neural circuits that mediate pupil-linked arousal remain unclear. The proposed project will test our central hypothesis: the cholinergic systems mediate pupil-linked arousal, in addition to the LC-NE system. Using a synthesis of optogenetic stimulation, genetic manipulation, and calcium imaging, we will test this hypothesis in two Aims. Aim 1 will determine the causal link between activation of the cholinergic systems and pupil size. Aim 2 will examine the extent to which cholinergic activity can be tracked by pupil size. This project will provide much-needed insight about the extent to which the cholinergic systems contribute to pupil-linked arousal. Such information is essential to rigorous and accurate interpretation of results from numerous studies using pupillometry, which is increasingly popularly used in cognitive neuroscience and psychiatry.