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Basic and translational (or preclinical) prostate cancer research has traditionally been conducted with a limited repertoire of immortalized cell lines, which have homogeneous phenotypes and have adapted to long-term tissue culture. Primary cell culture provides a model system that allows a broader spectrum of cell types from a greater number of patients to be studied, in the absence of artificially induced genetic mutations. Nevertheless, primary prostate epithelial cell culture can be technically challenging, even for laboratories experienced in immortalized cell culture. Therefore, we provide methods to isolate and culture primary epithelial cells directly from human prostate tissue. Initially, we describe the isolation of bulk epithelial cells from benign or tumor tissues. These cells have a predominantly basal/intermediate phenotype and co-express cytokeratin 8/18 and high molecular weight cytokeratins. Since prostatic stem cells play a major role in disease progression and are considered to be a therapeutic target, we also describe a prospective approach to specifically isolate prostatic basal cells that include both stem and transit-amplifying basal populations, which can be studied independently or subsequently differentiated to supply luminal cells. This approach allows the study of stem cells for the development of new therapeutics for prostate cancer.