Many sympathetic preganglionic neurons (SPN) in the intermediolateral cell column (IML) contain cocaine- and amphetamine-regulated transcript (CART), but the function of these CART-immunoreactive (IR) neurons is unknown. To test the possibility that CART might mark SPN involved in cardiovascular regulation, we first established whether all CART neurons in the spinal cord were SPN by double-immunofluorescent labelling for CART and choline acetyltransferase (ChAT). All autonomic subnuclei contained SPN immunoreactive for ChAT plus CART. Occasional ChAT-negative, CART-positive neurons occurred adjacent to the IML, indicating the existence of CART-IR interneurons. We then retrogradely labelled SPN with cholera toxin subunit B (CTB) from a variety of targets and used double immunofluorescence to detect CTB and CART. Among SPN in the IML, 43% projecting to the coeliac ganglion, 34% projecting to the major pelvic ganglion, and about 15% projecting to the superior cervical ganglion or adrenal medulla contained CART. CART also occurred in most SPN projecting to the major pelvic ganglion from either the central autonomic area (63%) or the intercalated nucleus (58%). Finally, we used drug-induced hypotension in conscious rats to evoke Fos immunoreactivity in barosensitive SPN and immunostained to reveal Fos and CART. CART immunoreactivity was present in 41% of the Fos-IR barosensitive neurons, which were concentrated in the IML of segments T5-T13. CART-positive, Fos-negative neurons also occurred in the same segments. These results indicate that CART occurs in barosensitive SPN, nonbarosensitive SPN, and interneurons. Thus, CART is not an exclusive marker for cardiovascular SPN but is likely to influence many autonomic activities.