The role of the brain renin-angiotensin system (RAS) in blood pressure (BP) and autonomic control isrecognized. However, the lack of the precise source of renin in the brain is one of the main barrierstowards a complete understanding of how the brain RAS operates. Here, we hypothesized that renin isexpressed in brain regions involved in sympathetic control such as the rostroventral lateral medulla(RVLM). We employed RNAscope, a highly sensitive and specific in situ hybridization technology.Surprisingly, the renin positive signals were absent in the RVLM, but we identified renin transcripts inthe nucleus ambiguus (NA), a site in the vicinity of the RVLM that contains cardiac vagal motoneurons.There was no signal in this region with the negative control scrambled probe. The signal strength wasgreater than that observed in other regions of the brain classically associated with the brain RAS. Wealso observed that the NA contains other necessary RAS components to produce angiotensin.Interestingly, renin was colocalized with cells expressing choline acetyltransferase (ChAT), suggesting itspossible role in cholinergic neurotransmission. Therefore, we generated mice lacking renin specifically incholinergic neurons (Ren-a ChAT-null ). Because we previously identified an alternative brain-specific reninisoform (ren-b) that elicits cardioprotective roles, we also generated mice lacking ren-b in cholinergicneurons (Ren-b ChAT-null ). At baseline, Ren-a ChAT-null exhibited a trend towards decreased BP, while Ren-b ChAT-null exhibited higher BP, although these differences were not statistically significant. Importantly,Ren-a ChAT-null exhibited an attenuated tachycardiac and pressor responses to atropine (p<0.005 andp<0.05, respectively; n=4-7), indicating a lower parasympathetic tone in these mice. Conversely, Ren-b ChAT-null exhibited an augmented pressor response to atropine (p<0.005; n=7) suggesting an elevatedparasympathetic tone. These data suggest that the activation of the brain RAS in the NA might play arole in cholinergic neurotransmission, and it defines the NA as a potential source of endogenous renin inthe brainstem. Studies to understand the differential contribution of Ren-a and Ren-b in the NA to BPare in progress.