Rotating Wall Vessel (RWV) Bioreactors simulate microgravity and facilitate 3-D tissue-like assembly through spatial co-localization and cell-cell interactions. This unique cell culture venue is well suited to assess the role of micro-environmental cues in the assembly and tissue-specific differentiation of cells in culture. Our long term goal is to use RWV Bioreactors for generating functional neuroendocrine 3-D constructs which may be useful as clinical replacement tissue in treating neurodegenerative diseases. As a model we are using PC12 pheochromocytoma cells, a bipotential rat adrenal medullary tumor cell line. PC 12 cells differentiate, depending on exogenous factors, either along the neuronal or the neuroendocrine pathway. PC12 cells, when maintained for up to 20 days in RWV Bioreactors, form macroscopic tissue-like aggregates which exhibit enhanced expression of neuroendocrine, adrenergic differentiation markers (Lelkes et al., In Vitro Devel. Biol, 1998, 34: 316–325). We hypothesized that exposure of PC12 cells to the “simulated microgravity” culture conditions in the RWV Bioreactors, might selectively activate signal transduction pathways leading to enhanced neuroendocrine adrenergic differentiation. Using quantitative RT-PCR we demonstrated rapid upregulation of an adrenergic marker, phenylethanolamine-N-methyl transferase (PNMT), in short term RWV cultures. Concomitantly, we found, by electrophoretic mobility shift assays, differential induction of nuclear transcription factors, such as GRE and SP-1, which are known to be involved in the glucocorticoid-induced activation of PNMT. Conversely, upon short term culture of PC12 cells in RWV, the neuronal traits of the cells were impaired. Upon exposure to simulated microgravity, MAPK signaling (erk and jnk) was constitutively activated, while nerve-growth factor (NGF)-induced activation of erk, was abrogated. These results suggest that the culture conditions in the RWV Bioreactors are sufficient to induce PC12 cell differentiation towards the neuroendocrine, phenotype by upregulating “adrenergic” gene expression, while downregulating neurotrophin signaling pathways.