Horseradish peroxidase (HRP) was used to determine whether neurons in the rat superior cervical ganglion (SCG) are localized in regions of the ganglion as a function of the postganglionic trunk they utilize. In separate experiments, each of the two major postganglionic trunks was cut 1-3 mm from the SCG and solid HRP was applied to the cut end proximal to the ganglion. The results demonstrated that the cell bodies of neurons whose axons project out the internal carotid nerve (ICN) were located primarily in the rostral part of the ganglion. Cell bodies of neurons whose axons project out the external carotid nerve (ECN) were located primarily in the caudal part. The total percentages of neuronc with axons in the ICN and ECN were about 35% and 45%, respectively. When HRP was applied to both these trunks, 73% of the neurons in the SCG were labeled. In the caudal portion of the ganglion, an additional group of neurons was observed whose axons project into the cervical sympathetic trunk. Control studies indicated that the neuronal labeling observed in our experiments was due to retrograde axonal transport rather than the direct uptake of HRP by neuronal cell bodies. Thus, neuronal subpopulations exist in specific regions of the rat SCG. The significance of these results to biochemical and electrophysiological studies is discussed.