Single cell electroporation (SCE) is an electrophysiological technique that uses trains of voltage pulses to disrupt the cell membrane and simultaneously drive macromolecules such as fluorescent dyes or DNA into a target cell. SCE has emerged as an important step in developing ‘connectomic’ technology that can resolve the architecture of neuronal networks in single cell resolution [1]. However, two key limitations restrict the applicability of SCE. Firstly, SCE requires direct visualisation of target neurons, limiting its use in vivo to regions of the brain that can be resolved optically. Secondly, SCE is incompatible with standard electrophysiological recording techniques, meaning that data from electroporated cells cannot be interpreted in light of their functional properties. In the current study we validate a novel technique that combines single cell electroporation with extracellular recording, permitting functional identification of neurons prior to electroporation with fluorescent dextrans or plasmids encoding a variety of genes. Methods: In vitro experiments were conducted on acute or cultured brain slices obtained from neonatal (P5-10) rat pups. Rats were deeply anaesthetised with isoflurane and killed by decapitation. In vivo experiments were conducted on urethane anaesthetised (acute experiments: dextran) or isoflurane anaesthetised (recovery experiments: plasmid) adult rats (300 – 500 g). Borosilicate pipettes filled with either fluorescent dextran or plasmid solutions were used to obtain extracellular recordings from single neurons. Once physical contact between pipette and cell was established the recording amplifier was programmed to deliver a train of current pulses through the pipette, using pipette resistance to calculate the current required to generate the desired voltage (7.5 V: dextran, -12 V: plasmid). Results: Dextran labelling was achieved in >95% of cells (N>40) recorded in vitro, provided the pipette was within 6 μm of the target cell. In vivo successful electroporation was obtained in 48/105 spontaneously active brainstem neurons recorded up to 9 mm deep . Preliminary experiments using plasmid DNA in the recording solution indicate that the same principle is compatible with single cell gene delivery: fluorescent proteins are transcribed within 18 hours of electroporation in cultured neurons and in vivo, although further refinements are required to improve the reliability of this approach. Conclusion: We have developed a technique that combines SCE with extracellular recording, negating the need for optical guidance and permitting functional identification of target neurons prior to electroporation. This technique will permit the application of novel genetic techniques to physiologically characterised neurons.