Neurturin and glial cell line-derived neurotrophic factor are novel mitogens for normal adult rat chromaffin cells in vitro. These neurotrophic factors differ from the previously described adult chromaffin cell mitogens, nerve growth factor and basic fibroblast growth factor, in that their effects are potentiated by depolarization and activation of protein kinase C. Neurturin and glial cell line-derived neurotrophic factor signal via the receptor tyrosine kinase, ret, but may also act independently of ret. Both depolarization and phorbol esters act synergistically with neurturin to up-regulate ret protein expression in chromaffin cell cultures, suggesting a mechanism for potentiation of mitogenesis. However, a direct role for ret in mitogenesis has not been established. Stimulation by neurturin causes increased phosphorylation of extracellular signal-regulated kinases 1 and 2 in cultured chromaffin cells, and mitogenesis is prevented by inhibitors of their phosphorylation. Inhibitors of phosphatidylinositol 3-kinase also prevent mitogenesis. The present findings suggest the hypothesis that neurotrophic factors and neurally derived signals might cooperatively regulate chromaffin cell proliferation in vivo in the rat. In addition, trans-synaptic stimulation might provide a route by which epigenetic factors could influence the development of adrenal medullary hyperplasia in humans with hereditary multiple endocrine neoplasia syndromes 2A and 2B by affecting expression and/or activation of ret.