Substance P (SP) is elevated in the intermediate zone caudal to a spinal cord lesion presumably due to sprouting of intraspinal and primary afferent axons. It is unclear, however, if axon terminals are in direct contact with preganglionic neurons located within the different autonomic subnuclei. Therefore, the innervation of preganglionic sympathetic neurons by SP was quantified using confocal imaging and morphometric image analysis. The number of SP-immunoreactive varicosities apposed to nitric oxide synthase-positive neurons significantly increased bilaterally in all sympathetic areas of segment T2 one week after low cervical hemisection at C6/7. Consequently, direct excitatory effects of SP on preganglionic neurons may play an important role in the dysregulation of arterial blood pressure observed in patients with spinal cord injury at the cervical or upper thoracic level.