In multipolar vertebrate neurons, action potentials (AP) initiate close to the soma, at the axonal initial segment (AIS). Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (NaV) channels are a functional hallmark of the AIS in vertebrates. We used an intronic MiMIC to determine the endogenous gene expression and subcellular localization of the sole NaV channel in both male and female Drosophila, para. Despite being the only NaV channel in the fly, we show that only 23 ±1% of neurons in the embryonic and larval CNS express para, while in the adult CNS para is broadly expressed. We generated a single-cell transcriptomic atlas of the whole 3rd instar larval brain to identify para expressing neurons and show that it positively correlates with markers of differentiated, actively firing neurons. Therefore only 23 ±1% of larval neurons may be capable of firing NaV-dependent APs. We then show that Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon, which we named the Distal Axonal Segment (DAS). The DAS is present in multiple neuron classes in both the 3rd instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Nav-dependent APs originate in the DAS. Identification of the distal NaV localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates.SIGNIFICANCE STATEMENTThe site of AP in invertebrates is unknown. We tagged the sole NaV channel in the fly, para, and identified that Para is enriched at a distal axonal segment (DAS). The DAS is located distal to where dendrites impinge on axons and is the likely site of AP initiation. Understanding where APs are initiated improves our ability to model neuronal activity and our interpretation of electrophysiological data. Additionally, para is only expressed in 23 ±1% of 3rd instar larval neurons but is broadly expressed in adults. Single-cell RNA sequencing of the 3rd instar larval brain shows that para expression correlates with the expression of active, differentiated neuronal markers. Therefore, only 23 ±1% of 3rd instar larval neurons may be able to actively fire NaV-dependent APs. Copyright © 2020 the authors.