The efficient delivery of foreign genes into postmitotic cells is becoming very important for studies of nervous system functions. Cultured sympathetic neurons synthesize neuropeptide Y (NPY) in addition to catecholamines, providing an experimental model for studying neuronal peptide biosynthesis. In this work, we have studied the biosynthetic processing of NPY in primary cultures of rat superior cervical ganglion (SCG) neurons. NPY activation is complex, requiring sequential actions of a prohormone convertase (PC), carboxypeptidase H, and peptidylglycine alpha-amidating mono-oxygenase. Northern analyses established that SCG neurons in the animal contain mRNAs for both PC1 and PC2, and simultaneous immunocytochemistry for NPY and PC1 or PC2 established a 1:1 correspondence between NPY and PC2 expression in two thirds of the neurons that express NPY, both in the animal and in tissue culture. Biosynthetic studies on proneuropeptide Y (pro-NPY) processing to mature NPY established a close similarity to the rates seen in endocrine cells expressing PC2 and established clear differences between the patterns in SCG neurons and in endocrine cells expressing PC1. Recombinant adenoviruses were used to increase the level of PC1 in the cultured neurons from negligible to a level comparable with the level of PC1 in the anterior pituitary, and pro-NPY processing was markedly accelerated. When the viruses were used to lower the endogenous PC2 levels, using an antisense construct, pro-NPY processing was retarded. Taken together, these results support a major role for PC2 as the pro-NPY converting enzyme, and they establish the cultured SCG neurons as a model to study neuronal peptide biosynthesis.