The sympathetic preganglionic neurons (SPN) of the intermediolateral cell column (IML) play a critical role in the maintenance of vascular tone. We undertook a comparative neuroanatomical analysis of neuronal nitric oxide synthase (nNOS) expression in the SPN of the mature normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rat (SHR). The anatomical relationship between nNOS and the NO signaling molecule cyclic guanosine monophosphate (cGMP) was also determined. All animals were male, age >6 months. Fluorogold (FG) retrograde labeling of SPN (detected with immunohistochemistry) was combined with NADPH-diaphorase histochemistry for NOS in the thoracic spinal cord (T1–11, n = 5 WKY, 5 SHR). There was no difference in the total number of FG-labeled SPN (WKY 6,542 ± 828, SHR 6,091 ± 820), but the proportion of FG-labeled cells expressing NOS was significantly less in the SHR (WKY 64.4 ± 5.1 vs. SHR 55.6 ± 2.1, P < 0.05). Fluorescence immunohistochemistry for nNOS/cGMP (n = 4 WKY, 4 SHR) was also performed. Confocal microscopy revealed that all nNOS-positive SPN contain cGMP and confirmed a strain-specific anatomical arrangement of SPN cell clusters. A novel subpopulation of cGMP-only cells were also identified. Double labeling for cGMP and choline acetyltransferase (n = 3 WKY, 3 SHR), confirmed these cells as SPN in both WKY and SHR. These results suggest that cGMP is a key signaling molecule in SPN, and that a reduced number of NOS neurons in the SHR may play a role in the increase in sympathetic tone associated with hypertension in these animals.