A protocol which was developed for the culture of epithelial cells from radical prostatectomy specimens was slightly modified to permit the culture of cells from ultrasound-guided prostatic needle biopsies. The collagenase digestion step of the standard protocol was omitted, and biopsies were simply minced and allowed to attach to collagen-coated dishes in serum-free medium. Cell outgrowths from biopsies were free of fibroblasts, and expression of keratin, prostate specific antigen, and prostatic acid phosphatase was maintained in vitro. The establishment of a bank of frozen cells from primary cultures permitted repetitive studies with individual cell strains, which could be serially passaged and were capable of clonal growth. The ability to derive cultures from biopsies will facilitate the biological characterization of cells from primary prostate tumors of high malignant grade, which are not commonly available from radical prostatectomy specimens.