Androgen ablation inhibits androgen receptor (AR) activity and is as an effective treatment for advanced prostate cancer (PCa). Invariably, PCa relapses in a form resistant to further hormonal manipulations. Although this stage of the disease is androgen-refractory, or androgen depletion-independent (ADI), most tumors remain AR-dependent through aberrant mechanisms of AR activation. We employed the LNCaP/C4-2 model of PCa progression to study AR activity in androgen-dependent and ADI PCa cells. In this report, we show that the AR is transcriptionally inactive in androgen-dependent LNCaP cells in the absence of androgens. However, in ADI C4-2 cells, the AR displays a high level of constitutive, androgen-independent transcriptional-activity. To study the me chanisms of ligand-dependent and ligand-independent AR activation in these AR-expressing cells, we generated a reporter system based on swapping the DNA binding domain of the AR with the DNA binding domain of the yeast Gal4 transcription factor. In androgen-dependent PCa cells, the well characterized C-terminal AR activation function-2 (AF-2) domain was critical for strong, ligand-dependent activity. Conversely, in ADI PCa cells, constitutive, ligand-independent AR activity was AF-2-independent but instead dependent on N-terminal AR domains. Importantly, the ligand- and AF-2-independent mode of AR activation observed in ADI PCa cells was completely resistant to the antiandrogen, bicalutamide. Our data thus demonstrate that the AR can inappropriately activate transcription in ADI PCa cells via mechanisms that are resistant to castration and AR antagonism, the two modes of androgen ablation used to treat advanced PCa.