Adult rat sympathetic neurons can possess specific neuropeptides utilized as cotransmitters along with norepinephrine, but the factors that regulate their expression remain unknown. 60% of adult rat superior cervical ganglion (SCG) neurons express neuropeptide Y (NPY) in vivo. To determine whether the restricted expression was an intrinsic property of sympathetic ganglia, we examined if embryonic sympathetic precursors gave rise to NPY immunoreactive (-IR) neurons in vitro. After one week in culture, 60% of neurons derived from the E14.5 rat SCG were NPY-IR. Thus, ganglia isolated before peripheral target contact or preganglionic innervation were capable of regulating NPY expression both in the number of neurons with NPY and in the developmental timing of NPY expression. To determine if the restricted expression of NPY was a reflection of neuroblasts committed to an NPY fate, SCG precursors were labeled with a replication incompetent retrovirus carrying lacZ, and NPY expression in lacZ-labeled clones examined after one week. Two thirds of neuronal clones obtained were uniformly NPY-IR; that is, all neurons in a clone either possessed or lacked NPY. One-third of the neuronal clones were mixed and contained both neurons with and without NPY. We provide a novel demonstration that both lineage and environmental cues contribute to neuropeptide phenotype.