Diseases of the prostate gland are the most frequently diagnosed in men after middle age. Spheroid cell culture is a useful technique for tissue engineering or regenerative medicine research, pharmacological and toxicological studies, and fundamental studies in cell biology. In this paper, we investigated zinc distribution in cell spheroids in benign prostate hyperplasia (BPH) and prostate cancer (PCa) (DU145) and analyzed the differences in the response to zinc (0-150 μM) treatment using μXRF with synchrotron radiation. The results showed nonuniform distribution of Zn in all the spheroids analyzed. The differential response to zinc of BPH and DU145 cell spheroids suggests that zinc may have an important role in PCa.