Control of the cardiovascular system is dependent upon a multisynaptic pathway that regulates sympathetic nerve activity. The paraventricular nucleus of the hypothalamus (PVN) and nucleus tractus solitarius (NTS) are critical components of the neural circuit that regulate sympathetic nerve activity3. The NTS projects to the PVN but there is no evidence to indicate this projection targets those preautonomic neurones in the PVN that regulate sympathetic activity. It is important to establish this as studies suggest elevated sympathetic nerve activity, strongly associated with cardiovascular disease including heart failure, is partly generated from the preautonomic neurones of the PVN3. The present study used anterograde (Biotin dextran amine-BDA) and retrograde (Fluorogold-FG or cholera toxin subunit B-CTB) labelling methods to identify the terminal neuronal targets of the ascending axonal projection from the NTS to the PVN. All experiments were performed in accordance with the Animals (Scientific Procedures) Act, 1986. Studies were performed on male Wistar rats (n=8, 280-310 g). For anterograde labelling of NTS projecting axons, animals were anaesthetised using a combination of medetomidine (0.25 ml/100g) and ketamine (0.06 ml/100g). Animals received either an iontophoretic deposit or pressure injection (10 nl) into the NTS of 10 % BDA using the coordinates R/C -13.68 mm, L 0.5 mm and D/V -8.00 mm2,4,5. After a recovery period (14 days) to allow anterograde transport of BDA to the PVN, animals were re-anaesthetised as previously described to allow an injection of either 1μl of 2% FG or 5μl of 0.5% CTB into the spinal cord to label spinally projecting preautonmic PVN neurones. Post-operatively during the recovery period, animal welfare was monitored with administration of analgesia (buprenophine 0.1 ml/kg) as required. Following a 7-14 day recovery period animals were humanely killed (pentobarbital 60 mg/kg) perfused fixed (4% paraformaldehyde-PFA or 4% PFA and 0.5% glutaraldehyde) with removal of brain and spinal cord. Frozen sections (40 μm) were processed to reveal BDA and CTB1,4. Tissues were examined under bright field or epifluorescence for the existence of putative connections between the NTS and the preautonomic neurones of the PVN. The combination of techniques revealed ascending axons from the NTS that coursed through and around the PVN nucleus. The NTS terminals showed numerous varicosities, some of which appeared to closely appose the somata and dendrites of preautonomic spinally projecting PVN neurones. This is the first demonstration that axonal fibres from the NTS target preautonomic neurones of the PVN. Functionally, this pathway has the potential to modulate the activity of those neurones controlling sympathetic outflow. It must also be considered that this pathway may contribute to the generation of abnormal sympathetic activity by the PVN.