Pituitary adenylate cyclase activating polypeptides (PACAP27 and PACAP38) are members of the VIP/secretin/glucagon family of peptides and have diverse neuroregulatory effects in sympathoadrenal cell development and function. PACAP peptides regulate rat superior cervical ganglion (SCG) neuron catecholamine and neuropeptide Y content and secretion, and promote sympathoneuroblast survival through activation of specific PACAP1 receptor isoforms. In examining the potential sources of PACAP regulating the SCG, PACAP expression was identified in rat preganglionic neurons in the intermediolateral cell column (IML) of the thoracic spinal cord which provide primary afferent projections to this sympathetic ganglion. Thoracic spinal cord segments (T1-4) contained approximately 17 pmol PACAP38 immunoreactivity/g tissue wet weight. Reverse-transcription polymerase chain reaction of cDNA from microdissected thoracic spinal cord using primers specific for rat neuronal proPACAP identified proPACAP mRNA expression in the IML; the results correlated with neurons labeled for proPACAP mRNA by in situ hybridization histochemistry and implicated PACAP biosynthesis in IML neurons. To demonstrate directly proPACAP transcript expression in preganglionic projection neurons to the SCG, the ganglion was decentralized and the sympathetic trunk immersed in fluorogold to identify sympathetic preganglionic neurons by retrograde labeling. Cryosections of spinal cord segments containing preganglionic neuron fluorogold labeled neurons were processed subsequently for in situ hybridization histochemical localization of proPACAP mRNA using a digoxigenin-labeled riboprobe; IML neurons were examined for fluorogold and digoxigenin/alkaline phosphatase product dual labeling. More than half of the preganglionic projection neurons to the SCG expressed PACAP mRNA, consistent with the postulate that PACAP peptides released from a subpopulation of thoracic IML preganglionic neurons may be physiological anterograde modulators of sympathetic SCG function.