Acetylcholine acts as a neurotransmitter/neuromodulator of many central nervous system processes such as learning and memory, attention, motor control, and sensory processing. The present study describes the spatial distribution of cholinergic neurons throughout the brain of the weakly electric fish, Apteronotus leptorhynchus, using in situ hybridization of choline acetyltransferase mRNA. Distinct groups of cholinergic cells were observed in the telencephalon, diencephalon, mesencephalon, and hindbrain. These included cholinergic cell groups typically identified in other vertebrate brains, for example, motor neurons. Using both in vitro and ex vivo neuronal tracing methods, we identified two new cholinergic connections leading to novel hypotheses on their functional significance. Projections to the nucleus praeeminentialis (nP) arise from isthmic nuclei, possibly including the nucleus lateralis valvulae (nLV) and the isthmic nucleus (nI). The nP is a central component of all electrosensory feedback pathways to the electrosensory lateral line lobe (ELL). We have previously shown that some neurons in nP, TS, and tectum express muscarinic receptors. We hypothesize Abbreviations: cELL, commissure of the electrosensory lateral line lobe; CC, crista cerebellaris; CCb, corpus cerebelli; CLS, centrolateral segment of the ELL; CMS, centromedial segment of the ELL; DD, dorsodorsal telencephalon; DFI, lateral subdivision of the diffuse nucleus of the inferior lobe; DL, dorsolateral telencephalon; DLv, dorsolateral telencephalon ventral subdivision; DM2v/d, dorsomedial division of the telencephalon (dorsal and ventral); Dp, dorsal posterior telencephalon; DPI, lateral subdivision of the caudal DP; DPm, medial subdivision of the caudal DP;