Cancer cells are heterogeneous in both their phenotypes and ability to promote tumor growth and spread. Xenografting is used to identify the most highly capable cells to regenerating tumors, referred to as cancer repopulating cells. Because prostate cancers rarely grow as xenografts, indentifying prostate cancer repopulating cells has not been possible. Here we report improved methods to xenograft localized primary prostate cancer tissues using chimeric grafts with neonatal mouse mesenchyme. Xenograft survival of tumor tissue was significantly increased by neonatal mesenchyme (6/6 patients 66 of grafts vs 4/6 patients 41 of grafts) and doubled the proliferation index of xenografted cancer cells. When applied to isolated prostate cancer cells, neonatal mesenchyme effectively reconstituted prostate cancers and increased xenograft survival (4/9 patients; 32 of grafts with mesenchyme, 0 without), and supported active cancer cell proliferation. Using this assay, we showed that unfractionated alpha2beta1integrin(hi) and alpha2beta1integrin(lo) cells from primary localized prostate cancers demonstrated tumor formation at comparable rates, similar to previous reports using metastatic specimens. Thus, this new protocol efficiently established tumors and enabled proliferative expansion of both intact tumor tissue and fractionated cancer cells, providing a bioassay to identify and therapeutically target prostate cancer repopulating cells.