Dehydroepiandrosterone sulfate is classically seen as an inactive reservoir for the production of dehydroepiandrosterone. Steroid sulfatase is the enzyme that catalyzes the hydrolysis of dehydroepiandrosterone sulfate to dehydroepiandrosterone, which can then be further metabolized to other steroid hormones. Recent studies, however, indicate that dehydroepiandrosterone sulfate can mediate biological effects without being converted to dehydroepiandrosterone. This study aims to evaluate whether dehydroepiandrosterone sulfate itself influences the differentiation of PC-12 cells or if its desulfation to dehydroepiandrosterone is required. dehydroepiandrosterone and dehydroepiandrosterone sulfate both influence the differentiation of chromaffin PC-12 cells. Blocking steroid sulfatase activity and thereby the conversion of dehydroepiandrosterone sulfate to dehydroepiandrosterone by the enzyme blocker estrone sulfamate showed that the effect of dehydroepiandrosterone sulfate is independent of its conversion to dehydroepiandrosterone. Dehydroepiandrosterone sulfate, similar to dehydroepiandrosterone, reduced nerve growth factor-induced neurite outgrowth of PC-12 cells and the expression of synaptosomal-associated membrane protein of 25. kDa, increased the expression of chromogranin A and significantly increased dopamine release of PC-12 cells. In addition, dehydroepiandrosterone sulfate, dehydroepiandrosterone and membrane impermeable dehydroepiandrosterone-BSA all significantly reduced NGF-induced MAPK ERK1/2 signaling after 5. min. In summary, this study provides evidence that dehydroepiandrosterone sulfate, independent of its conversion to dehydroepiandrosterone, directs PC-12 cells' differentiation to a neuroendocrine direction. Furthermore, employing membrane-impermeable dehydroepiandrosterone-BSA indicates the involvement of plasma-membrane bound receptors.