Although some sensory ganglion cells in mature animals are catecholaminergic, most mammalian sensory neurons that express the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) do so only transiently during early gangliogenesis in vivo. The lack of TH expression at later stages appears to be due to modulation of this catecholaminergic potential. A previous study showed that the phenotype reappears, for example, when E16.5 and older sensory ganglia are dissociated in culture into single cells, suggesting that extracellular influences can modulate TH expression. Moreover, TH expression in dissociate cultures is cell-density dependent, as a four-fold increase in plating density led to a 30% decrease in the percentage of TH neurons. The present study demonstrates that inhibition of TH expression in high density cultures is mediated by ganglionic non-neuronal cells (NNC), as removal of NNC abolished density-dependent inhibition. Moreover, plating E16.5 trigeminal neurons at low density on top of NNC monolayers resulted in an 85% decrease in the percentage of TH neurons. Treatment of cultures with non-neuronal cell conditioned medium (NNC-CM) reproduced the effect of coculture with NNC, suggesting that diffusible factors from NNC were involved in the inhibition of TH. The inhibitory effect of NNC-CM was mimicked by treatment of dissociate cultures with ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF). However, immunoprecipitation of NNC-CM with antibodies against LIF or CNTF showed that only anti-LIF antibodies were able partially to remove the TH inhibitory activity of NNC-CM. Therefore, LIF is one, but not the only, factor mediating NNC inhibition of TH expression in cultured sensory neurons. In summary, these data indicate that ganglionic NNC can regulate sensory transmitter phenotype in culture by inhibiting expression of specific molecular traits. The finding that LIF can partially account for the inhibitory effect of ganglionic NNC on TH expression suggests a novel role for this cytokine in regulating differentiation of catecholaminergic properties in sensory neurons.