Androgens are potent differentiation agents that regulate prostate-specific antigen (PSA) gene expression via the androgen receptor (AR) that binds to androgen response elements (AREs) on the PSA gene to initiate transcription. However, in the absence of androgens, PSA gene expression can become elevated. This suggests that either the AR can be activated in the absence of androgen to elevate PSA gene expression through AREs on the PSA gene or that another transcription factor acting on the PSA promoter is stimulated. We have previously shown in vivo that butyrate, a differentiation agent that causes cell cycle arrest, increases serum PSA levels in castrated animals. Therefore, to determine the mechanism of butyrate induction of PSA, we used the LNCaP human prostate cancer cell line. Northern analyses and transfection experiments using a PSA reporter plasmid demonstrated induction of PSA gene expression by butyrate in LNCaP cells. Application of the antiandrogen bicalutamide blocked the induction of PSA mRNA by butyrate, suggesting a mechanism dependent on the AR. Consistent with this conclusion, electromobility shift assays showed increased AR-ARE complex formation with nuclear extracts from butyrate-treated cells. In addition, other reporter gene constructs that contain AREs were also induced by butyrate. Western blot analysis showed an increase in nuclear levels of AR protein in cells exposed to butyrate, whereas whole cell levels remained unchanged, suggesting that butyrate causes nuclear translocation of the AR. Thus, the differentiation agent butyrate causes ligand-independent activation of the AR to increase expression of the differentiation marker PSA in human prostate cancer cells.