Mechanisms regulating peptide neurotransmitter metabolism were examined in dissociated cell cultures of the neonatal rat superior cervical ganglion (SCG). The pineal gland, a target of the SCG, produced a soluble factor (PCM) which increased substance P (SP) levels more than 15-fold in sympathetic neurons cultured in the presence of ganglion non-neuronal cells. Elimination of the non-neuronal cells decreased SP to negligible levels and abolished the stimulatory effects of PCM on SP expression. These observations suggest that ganglion non-neuronal cells stimulate sympathetic expression of SP, and that the pineal influences neuronal SP by acting on, or in concert with, ganglion support cells. PCM also influenced other neurotransmitter systems. In the presence of ganglion non-neuronal cells, PCM treatment increased choline acetyltransferase (CHAC) and decreased tyrosine hydroxylase (TOH) and somatostatin (SO). By contrast, PCM treatment of pure neuronal cultures resulted in negligible CHAC and SP levels and a doubling of SO with a small increase in TOH. In sympathetic neurons, SP expression may be associated with cholinergic development, whereas SO may be associated with noradrenergic phenotypic expression. Moreover, there is a reciprocal relationship between SP and SS expression by sympathetic neurons analogous to the previously described relationship between noradrenergic and cholinergic expression.