An osmotic challenge elicits a sympathoexcitatory response that involves hypothalamic projections to the intermediolateral cell column (IML) of the spinal cord [1]. Moreover, fibres immunoreactive for vasopressin make up an abundant terminal network in the IML [2], which has been associated with sympathoexcitation [3,4]. Using a novel in situ rat preparation, we sought to investigate whether hypothalamic vasopressinergic spinally projecting neurones are activated during acute increases in plasma osmolality to elicit sympathoexcitation. Male Wistar rats (70-90 g) were anaesthetised deeply with halothane (5%) and decorticated, which included removal of the thalamus but preserved hypothalamic structures. Animals were arterially perfused with Ringer solution containing Ficoll (1.25%) at 31°C. Via a suction electrode lumbar sympathetic nerve activity (LSNA) was monitored while switching the perfusate from being isosmotic (291±1 mosmol (kg water)^–1) to hyperosmotic (321±1 mosmol (kg water)^–1) over 40s. For intrathecal injections, a calibrated glass micropipette was positioned into the subarachnoid space and a volume of up to 0.5 μl was delivered. Initially, we evaluated the response of intrathecal injections of a V₁ receptor agonist (Phe²,Ile³,Orn⁸-Vasopressin; 1 μg ml^–1) on LSNA before and after intrathecal injections of a V_1a receptor antagonist (β-mercapto-β-β-cyclopentamethylenepropionyl¹,O-me-Tyr²,-Arg⁸-Vasopressin; 1 μg ml^–1). The V₁ receptor agonist induced sympathoexcitation (18±1%; n=3). This increase in LSNA was attenuated significantly by prior intrathecal delivery of a V_1a receptor antagonist to 6±2% (P<0.05, n=3). This dose of antagonist was then used to assess the hyperosmotic perfusate induced increase in LSNA. In control, hyperosmotic perfusate increased LSNA by 29±6% (n=5) but this was attenuated reversibly to 4±1% (P<0.001, n=5) at 2 min after intrathecal injection of the V_1a receptor antagonist (1 μg ml^–1). Thirty minutes later the hyperosmotic-induced increase in LSNA had recovered to control levels (25±3%). Our findings demonstrate the functional importance of the spinally projecting vasopressinergic pathway from the hypothalamus. This acts to stimulate spinal V_1a receptors and mediate the sympathoexcitation following acute salt loading.