We previously showed that prostatic stem cells are concentrated in the proximal regions of prostatic ducts. We now report that these stem cells can be purified from isolated proximal duct regions by virtue of their high expression of the cell surface protein stem cell antigen 1 (Sca-1). In an in vivo prostate reconstitution assay, the purified Sca-1-expressing cell population isolated from the proximal region of ducts was more effective in generating prostatic tissue than a comparable population of Sca-1-depleted cells (203.0 ± 83.1 mg vs. 11.9 ± 9.2 mg) or a population of Sca-1-expressing cells isolated from the remaining regions of ducts (transit-amplifying cells) (31.9 ± 24.1 mg). Almost all of the proliferative capacity of the proximal duct Sca-1-expressing cell population resides within the fraction of cells that express high levels of Sca-1 (top one-third), with the proximal region of prostatic ducts containing 7.2-fold more Sca-1^high cells than the remaining regions. More than 60% of the high-expressing cells coexpress α6 integrin and the antiapoptotic factor Bcl-2, markers that are also characteristic of stem cells of other origins. Further stratification of the phenotype of the stem cells may enable the development of rational therapies for treating prostate cancer and benign prostatic hyperplasia.