Transmission from striatal cholinergic interneurons (CINs) controls dopamine release through nicotinic acetylcholine receptors (nAChRs) on dopaminergic axons. Anatomical studies suggest that cholinergic terminals signal predominantly through non-synaptic volume transmission. However, the influence of cholinergic transmission on electrical signaling in axons remains unclear. We examined axo-axonal transmission from CINs onto dopaminergic axons using perforated-patch recordings, which revealed rapid spontaneous EPSPs with properties characteristic of fast synapses. Pharmacology showed that axonal EPSPs (axEPSPs) were mediated primarily by high-affinity α6-containing receptors. Remarkably, axEPSPs triggered spontaneous action potentials, suggesting that these axons perform integration to convert synaptic input into spiking, a function associated with somatodendritic compartments. We investigated the cross-species validity of cholinergic axo-axonal transmission by recording dopaminergic axons in macaque putamen and found similar axEPSPs. Thus, we reveal that synaptic-like neurotransmission underlies cholinergic signaling onto dopaminergic axons, supporting the idea that striatal dopamine release can occur independently of somatic firing to provide distinct signaling. Display omitted •Cholinergic interneurons signal to DA axons with phasic axonal EPSPs (axEPSPs)•AxEPSPs have many characteristics similar to synaptic depolarizations•Spontaneous axEPSPs can evoke spontaneous action potentials locally in the axon•AxEPSPs shape input-output function of DA axons, distinct from somatic signaling Dopaminergic axons express nicotinic receptors that modulate and evoke neurotransmitter release. Using direct recordings from dopaminergic axons, Kramer et al. reveal the presence of spontaneous synaptic-like nicotinic axonal EPSPs capable of generating spontaneous axonal action potentials. These findings demonstrate the mechanisms underlying nicotinic-receptor-mediated membrane-potential signaling in dopaminergic axons.