We established adrenal medullary cell lines from transgenic mice expressing an oncogene, the temperature-sensitive simian virus 40 large T-antigen, under the control of the tyrosine hydroxylase promoter. A clonal cell line, named tsAM5D, conditionally grew at a permissive temperature of 33°C and exhibited the dopaminergic chromaffin cell phenotype as exemplified by the expression pattern of mRNA for catecholamine synthesizing-enzymes and secretory vesicle-associated proteins. tsAM5D cells proliferated at the permissive temperature in response to glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). At a nonpermissive temperature of 39°C, GDNF and CNTF acted synergistically to differentiate tsAM5D cells into neuron-like cells. In addition, tsAM5D cells caused to differentiate by GDNF plus CNTF at 39°C became dependent solely on nerve growth factor for their survival and showed markedly enhanced neurite outgrowth. In the presence of GDNF plus CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal marker genes including microtubule-associated protein 2, neuron-specific enolase, neurofilament, and growth-associated protein-43, indicating that the cells underwent neuronal differentiation. Thus, we demonstrated that tsAM5D cells could proliferate at permissive 33°C, and also had the capacity to terminally differentiate into neuron-like cells in response to GDNF plus CNTF when the oncogene was inactivated by shifting the temperature to nonpermissive 39°C. These results suggest that tsAM5D cells should be a good tool to allow a detailed study of mechanisms regulating neuronal differentiation.