The proportion of sympathetic preganglionic neurons (SPN) showing nitric oxide synthase (NOS) immunoreactivity appears to vary with innervation target and blood pressure level. For normotensive Sprague-Dawley rats (SD), we evaluated peroxidase immunolabelling for choline acetyltransferase (ChAT) plus NOS in spinal cord segments T1–L2 and assessed NOS immunofluorescence in SPN retrogradely labelled with cholera toxin B subunit from the adrenal medulla (AM) or superior cervical (SCG), coeliac (CG), or major pelvic (MPG) ganglia. We also compared the distributions and numbers of NOS-positive and NOS-negative/ChAT-positive lateral horn neurons in SD with those in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). In SD, WKY, and SHR, rostrocaudal, dorsoventral, and mediolateral differences occurred in the distributions of NOS-positive and NOS-negative/ChAT-positive neurons in the intermediolateral cell column (IML), whereas the two groups were similarly distributed throughout the central autonomic area (CAA). Among the four retrogradely labelled populations of SPN, the percentages showing NOS immunoreactivity differed (CG-projecting, 54.8% ± 0.7%; SCG-projecting, 75.3% ± 1.2%; MPG-projecting, 89% ± 1.1% and AM-projecting, 98.6% ± 0.2%). Within each retrogradely labelled group of SPN, the NOS-positive proportion also varied with subnuclear location (e.g., 25.5% ± 4.0% of CG-projecting SPN in the CAA vs. 82.7% ± 7.6% of CG-projecting SPN in the dorsolateral funiculus). The numbers of NOS-positive and NOS-negative/ChAT-positive neurons in T9–T11 were the same in SD and SHR but differed in WKY. Our results show that the expression of NOS within SPN varies depending on the target that they innervate and also on their subnuclear location. Our data indicate that there are no anatomical differences between nitric oxide-synthesizing SPN in normotensive SD and hypertensive SHR. J. Comp. Neurol. 514:492–506, 2009. © 2009 Wiley-Liss, Inc.