Establishment of transmitter phenotype is an essential step in neuronal development. Studies on rat sympathetic neurons both in vivo and in vitro have provided evidence that mature cholinergic sympathetic neurons arise from previously noradrenergic neurons. Cultured rat superior cervical ganglion neurons can be influenced by their environment to remain noradrenergic, to acquire dual transmitter function, or to become predominantly cholinergic. Several other neuronal traits, such as a variety of surface molecules and released proteins, change simultaneously with levels of catecholamine and acetylcholine production, suggesting that various components of transmitter phenotype are regulated in concert. In this report, tyrosine hydroxylase levels are compared in neurons cultured under noradrenergic, dual function, or cholinergic conditions. Both enzyme activity in cell extracts and immunocytochemical staining were measured. These methods showed significantly less tyrosine hydroxylase enzyme activity and immunoreactive material in cholinergic cultures compared to noradrenergic and dual function cultures. These data support the interpretation that a switch in transmitter status from noradrenergic to cholinergic has occurred. This interpretation contrasts with that of Iacovitti et al. (Iacovitti, L., T. H. Joh, D. H. Park, and R. P. Bunge (1981) J. Neurosci. 1: 685-690), who conducted their experiments under critically different culture conditions.