Nerve growth factor (NGF) is a polypeptide growth factor with specific trophic function in nerve cells and was initially investigated for its role as a key player in the regulation of peripheral innervations. The aim of this study was to examine the NGF-induced transdifferentiation of adrenal medullary cells, and to screen the major candidate differentially expressed proteins involved in the transdifferentiation. NGF was used to treat primary cultures of neonatal calf adrenal medullary cells and the effects of transdifferentiation were determined in association with cellular morphology, ultrastructure and changes in endocrine function. Differentially expressed proteins were screened and identified through two-dimensional gel electrophoresis and mass spectrometry. The protein spots showing differential expression were verified by western blot analysis. We observed neurite outgrowth in the adrenal medullary cells treated with NGF under a phase contrast microscope. Ultrastructure analysis revealed that there were rich drumstick-like and villiform processes on the cell membranes and vesicles were formed near the cell membranes. The cytoplasm was rich in mitochondria and the secretion of epinephrine was decreased. Two-dimensional gel electrophoresis revealed that among the differentially expressed proteins, 48 protein spots showed an upregulated expression and 37 protein spots showed a downregulated expression, and no 'all-or-none' spots with significant differences in expression were found. Fourteen protein spots with an upregulated expression and 6 with a downregulated expression were randomly selected for identification by mass spectrometry. Western blot analysis revealed that ras homologus oncogene (Rho) GDP dissociation inhibitor α (RhoGDIα) protein expression was significantly downregulated and peripherin protein expression was significantly upregulated. In brief, our data demonstrate that NGF can induce the differentiation of adrenal medullary cells into neurons, and that RhoGDIα and peripherin may play important roles in this process.