Mature sympathetic neurons contain one or more neuropeptides in addition to a classical neurotransmitter. We compared the development of two peptides, neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP), in rat superior cervical (SCG) and stellate ganglia. NPY immunoreactivity (-IR) was first detected at embryonic day (E) 12.5. It was of similar immunofluorescence intensity in almost all tyrosine hydroxylase (TH)-IR cells. In contrast, VIP-IR, of variable fluorescence intensity, appeared at E14.5 in a subset of TH-IR cells in the stellate ganglion but not in SCG. Both peptides were present in bromodeoxyuridine-labeled neuronal precursors as well as neurons. The intensity of NPY immunofluorescence increased until E16.5. Subsequently, while it continued to increase in some neurons, the intensity decreased in others so that at birth approximately 55% of SCG and stellate neurons were NPY-IR. Developmental changes in NPY concentration, determined by radioimmunoassay, were similar in both ganglia, increasing between E14.5 and E16.5 and then decreasing 60% between E16.5 and birth. VIP expression differed from that of NPY. The proportion of VIP-IR cells began to decrease the day after VIP-IR was first detected. Although VIP-IR was present in one-third of E14.5 TH-IR stellate cells, at birth only 2% were VIP-IR. VIP-IR, measured by radioimmunoassay, was uniformly severalfold more concentrated in the stellate than SCG, and its concentration decreased throughout embryonic development, 40% between E14.5 and E16.5 and 95% by birth. In situ hybridization revealed detectable mRNA for both NPY and VIP at E14.5 in stellate ganglion and mRNA for NPY, but not VIP, in SCG. Initially, ganglionic neuropeptide mRNA appeared uniformly distributed but became heterogeneous. Our data indicate that features of the diverse peptidergic phenotypes expressed by sympathetic neurons are present when peptides are first detected while others arise subsequently. The final acquisition of peptidergic phenotypic diversity is complex, entailing both early induction in many cells and subsequent restriction to specific subpopulations.