Abstract Acetylcholine is a neurotransmitter that plays a role in many aspects of hearing, including selective attention, learning, frequency selectivity, sound localization, and discrimination of speech sounds. It also plays a critical role in helping the brain adapt during normal development, during aging and in response to damage of the ear or central nervous system. Top-down modulation, in which higher brain centers modify early acoustic processing, contributes to many of these functions, often through interactions with cholinergic pathways. A long-term goal of this research is to understand how cholinergic inputs modulate brainstem auditory processing and how projections from auditory cortex contribute to these functions. The present proposal will address four main gaps in our knowledge that limit our understanding of cholinergic function in the auditory brainstem. The first gap in our knowledge is lack of information on sources of cholinergic input to most brainstem auditory nuclei. Aim 1 will identify these sources by using recently developed viral vectors in newly created transgenic mice. A second gap concerns divergent projections from individual cholinergic cells. Modulatory cells in other brain areas typically have branching axons to allow widespread modulation. Aim 2 will use multi-label tracing in transgenic mice to identify divergent circuits that could support concurrent modulation of large expanses of the auditory brainstem. A third gap is a nearly total lack of information on cholinergic receptor types associated with specific brainstem circuits. Numerous receptor types occur in the subcortical auditory system, suggesting a variety of short- and long-term effects. Aim 3 will combine anatomical tract tracing with fluorescent in situ hybridization (FISH) to identify specific cholinergic receptor components associated with particular ascending and descending auditory circuits. The fourth gap in our knowledge concerns the identity of brainstem cholinergic circuits that are directly contacted by projections from the auditory cortex. Aim 4 will use conventional tracers as well as trans-synaptic intersectional viral methods in transgenic animals to test the hypothesis that cortical projections contact cholinergic cells that innervate many subcortical auditory nuclei. These results will identify the cholinergic circuits that are responsible for cortically-driven release of acetylcholine. Overall, the results from the four Aims will provide fundamental information about brainstem cholinergic circuits, their targets within the auditory pathway, and the extent to which they may be activated by projections from higher brain centers. This information is essential for the design and interpretation of future experiments to understand cholinergic and top-down modulation and for insight into therapeutic approaches to prevent or treat deficits associated with dysfunction of these systems.