Collagenase, trypsin, and pronase were used in separate, parallel experiments to obtain cell suspensions from human prostates obtained from surgical resections and autopsies. All of the examined prostates demonstrated benign hyperplasia. The dissociation using pronase gave both the largest number of nucleated cells and the largest proportion of viable cells from prostates. Surgically resected prostates gave a larger number of cells per gram of tissue than prostates obtained from autopsy. Cells obtained from surgically resected prostates were separated both by isopycnic sedimentation and by velocity sedimentation in a previously described isokinetic gradient. The authors studied fifteen prostates obtained at surgery; using pronase, they obtained 2.1±3.5x106 cells/g. Of these cells, 34.0±14.7% contained histochemically demonstrable acid phosphatase. Cells from six prostates were separated by velocity sedimentation in the isokinetic gradients; in the purest fractions from the isokinetic gradients, 81.0±12.2% of nucleated cells had histochemically demonstrable acid phosphatase. The cells in the purest fractions appeared to be epithelial cells. More than 99% of these separated cells excluded trypan blue. Isopycnic sedimentation was not an effective means of purifying epithelial cells from human prostates.