ABSTRACT Depolarisation of distal axons is necessary for somatic action potentials to be translated into axonal neurotransmitter release. Here, we show that activation of striatal cholinergic interneurons (ChIs) and nicotinic receptors (nAChRs) on mouse DA axons transiently prevents the release of dopamine (DA) by subsequent stimuli for ~100 ms. Previous studies have shown that nAChRs on DA axons can drive ectopic action potentials in DA axons to trigger DA release. We demonstrate ex vivo that a lower level of activation of ChIs is needed to suppress DA release than to trigger it, an effect that is not due to DA depletion, but to restricted re-activation of DA axons. This axonal brake on DA output is stronger and more persistent in dorsal than ventral striatum. In vivo, we reveal a predominant depression of DA release by endogenous acetylcholine, as antagonism of nAChRs in dorsal striatum conversely elevated tonic DA detected with optic-fibre photometry of GRABDA2m sensor and promoted conditioned place-preference. Our findings reveal that ChIs acting via nAChRs limit activation of DA axons by subsequent DA neuron activity, uncoupling DA axons from ascending action potentials and generating a dynamic inverse scaling of DA release according to ChI activity. FILE DESCRIPTIONS This repository contains the following files: Key Resources Table (.xlsx) - Table containing details on key resources (antibodies, mouse lines, virus strains, and software), and the persistent identifiers for protocols and code used and generated in this study. Source Data Folder (.zip): _README_Source_Data (.txt) with detailed information about each dataset. Individual tabular datasets corresponding to each panel shown in the Main Figures 1 to 6 (.csv). Excel spreadsheet containing all tabular datasets plotted in Main Figures 1 to 6 (.xlsx) Supplementary Data Folder (.zip): _README_Supplementary_Data (.txt) with detailed information about each dataset. Individual tabular datasets corresponding to each panel shown in the Supplementary Figures (.csv). Excel spreadsheet containing all tabular datasets plotted in Supplementary Figures (.xlsx)