The androgen receptor (AR) is required for prostate cancer development and contributes to tumor progression after remission in response to androgen deprivation therapy. Epidermal growth factor (EGF) increases AR transcriptional activity at low levels of androgen in the CWR-R1 prostate cancer cell line derived from the castration-recurrent CWR22 prostate cancer xenograft. Here we report that knockdown of AR decreases EGF stimulation of prostate cancer cell growth and demonstrate a mechanistic link between EGF and AR signaling. The EGF-induced increase in AR transcriptional activity is dependent on phosphorylation at mitogen-activated protein kinase consensus site Ser-515 in the AR NH(2)-terminal region and at protein kinase C consensus site Ser-578 in the AR DNA binding domain. Phosphorylation at these sites alters the nuclear-cytoplasmic shuttling of AR and AR interaction with the Ku-70/80 regulatory subunits of DNA-dependent protein kinase. Abolishing AR Ser-578 phosphorylation by introducing an S578A mutation eliminates the AR transcriptional response to EGF and increases both AR binding of Ku-70/80 and nuclear retention of AR in association with hyperphosphorylation of AR Ser-515. The results support a model in which AR transcriptional activity increases castration-recurrent prostate cancer cell growth in response to EGF by site-specific serine phosphorylation that regulates nuclear-cytoplasmic shuttling through interactions with the Ku-70/80 regulatory complex.