The neuronal endoplasmic reticulum (ER) is an extensive, complex endomembrane system, containing channels and receptors that permit its lumen to act as a dynamic calcium store. In response to electrical or chemical stimulation, ER calcium can be released under strict spatiotemporal control to regulate processes ranging from synaptic plasticity to growth cone guidance (1). Currently, there is much controversy over the nature of the ER in neurons and how this intersects with calcium signalling. We hypothesise that in neurons the ER is a dynamic structure with a single continuous lumen. To test this notion and define the relationship between ER-dynamics and calcium signalling in healthy and diseased neurons, we engineered a novel ER-targeted GFP construct, termed LV-pIN-KDEL, (derived from our pIN-G trafficking reporter, 2), in a 3rd generation replication-defective, self-inactivating lentiviral vector system capable of mediating gene transduction in diverse dividing and non-dividing mammalian cells, including neurons. Following its expression in HEK293 (or COS-7) cells, LV-pIN-KDEL (or its parent pIN-KDEL) yielded a pattern of fluorescence that co-localised exclusively with the ER marker sec61β but no other major organelles. We found no evidence for cytotoxicity and only rarely inclusion body formation. To explore the utility of the probe in resolving the ER in live cells, HEK293 or COS-7 cells were transduced with LV-pIN-KDEL and, after 48h, imaged directly at intervals from 1min to several hours. LV-pIN-KDEL fluorescence reveals the endoplasmic reticulum as a tubular lattice structure whose morphology can change markedly within seconds. Although GFP can be phototoxic, the integrity of the cells and ER is retained even after light exposure for periods up to 24h. Using LV-pIN-KDEL we have imaged the ER in diverse fixed neuronal cultures and, using real-time imaging, found evidence for extensive, dynamic, remodelling of the neuronal ER in live hippocampal cultures and brain slices. Supported by BBSRC (UK).