Progression to hormone-refractory growth of prostate cancer has been suggested to be mediated by androgen receptor (AR) gene alterations. We analyzed AR for mutations and amplifications in 21 locally recurrent prostate carcinomas treated with orchiectomy, estrogens, or a combination of orchiectomy and estramustine phosphate using fluorescence in situ hybridization, single-strand conformation polymorphism, and DNA sequence analyses. Amplification was observed in 4 of 16 (25%) and amino acid changing mutations was observed in 7 of 21 (33%) of the tumors, respectively. Two (50%) tumors with AR amplification also had missense mutation of the gene. Four of five (80%) cancers that were treated with a combination of orchiectomy and estramustine phosphate had a mutation clustered at codons 514 to 533 in the N-terminal domain of AR. In functional studies, these mutations did not render AR more sensitive to testosterone, dihydrotestosterone, androstenedione, or beta-estradiol. Tumors treated by orchiectomy had mutations predominantly in the ligand-binding domain. In summary, we found molecular alterations of AR in more than half of the prostate carcinomas that recurred locally. Some tumors developed both aberrations, possibly enhancing the cancer cell to respond efficiently to low levels of androgens. Furthermore, localization of point mutations in AR seems to be influenced by the type of treatment.