The major source for dehydroepiandrosterone (DHEA) and its sulphate compound DHEA-S is the inner zone of the adrenal cortex, which is in direct contact to adrenomedullary chromaffin cells. Due to their close proximity, direct interactions of DHEA and DHEA-S with chromaffin cells during adrenal gland development and throughout the whole life span are hypothesized. A possible direct effect of DHEA-S and the cellular and molecular mechanisms of DHEA-S action on chromaffin cells remain unresolved. Therefore, in this study, we aimed at clarifying DHEA-S effects and mechanisms of action on rat chromaffin PC12 cells. DHEA-S (10^-6 mol/l) inhibited nerve growth factor (NGF, 20 ng/ml)-induced cell proliferation by 66% (n = 4, p < 0.001). In NGF-stimulated cells, neuronal differentiation was inhibited by DHEA-S, as demonstrated by a 22% reduction (n = 3; p < 0.05) of neuronal differentiation marker expression, synaptosome-associated protein of 25 kDa (SNAP-25), and a 59% (n = 6; p < 0.001) decrease in neurite outgrowth. Moreover, DHEA-S stimulated expression of endocrine marker chromogranin A (CgA) by 31% (n = 4; p < 0.05 vs. control) and catecholamine release from NGF-treated PC12 cells by 229% (n = 3-5; p < 0.001), indicating a DHEA-S-induced shift towards neuroendocrine differentiation. On a molecular level, DHEA-S diminished NGF-induced ERK1/2 phosphorylation. Taken together, DHEA-S inhibited NGF-induced proliferation and neuronal differentiation and shifted cells towards a more endocrine phenotype. Interference of DHEA-S with NGF-stimulated ERK1/2 activation might be involved in this effect. Our study provides support for the notion that adrenocortical-derived DHEA-S impacts adrenomedullary chromaffin cells during development and differentiation.