Neuroendocrine (NE) cells are enigmatically found in association with human prostate cancers and their numbers are reported to increase in advanced and hormoneresistant tumors. The origin of this cell type and the reason for their appearance in prostate tumors remains unresolved. Previously, Bang et al. (Proc Natl Acad Sci USA 1994;91:5330) reported that dibutyryl adenosine 3',5'-cyclic phosphate (db-cAMP), an agent that upregulates intracellular cAMP, was able to induce a NE cell-like phenotype of cultured human prostate cancer cells, including the androgen-sensitive LNCaP line. Here we report that chronic incubation of LNCaP cells in a medium containing 10% charcoal-stripped fetal bovine serum (CSFBS) likewise induces NE differentiation of these cells. Within 5 days of switching low density cultures of LNCaP cells to this modified medium, the cells growth arrest and acquire an altered morphology with numerous cytoplasmic secretory granules and elongated processes that resemble cultured neurons. This morphology predominates at 10 days with complete transformation seen by 20 days of culture. Electron microscopic analysis of sections of CS-FBS maintained cells showed the presence of abundant dense core secretory granules characteristic of NE cells. Immunohistochemical staining identified the upregulation of the expression of NE markers bombesin, neuron-specific enolase, and S-100 in this modified culture medium. Once established, the NE cell-like phenotype was found to be reversible upon replacement with a medium containing unmodified fetal bovine serum, but not by direct supplementation of CS-FBS medium with dihydrotestosterone (DHT) (I nM). DHT supplementation did, however, suppress the development of the NE cell-like phenotype when it was present at the initiation of exposure to CS-FBS medium. In contrast to db-cAMP treatment, which did not affect prostate specific antigen (PSA) or androgen receptor (AR) expression of LNCaP cells, NE-differentiated LNCaP cells derived in this hormone-deficient medium showed marked downregulation of PSA and AR expression. These in vitro results further support the concept that prostate cancer cells can tranform in vivo to cells with a NE phenotype and suggest that this transformation might be accelerated in patients by certain therapies for prostate cancer.