a Cancer is a worldwide public health problem, and its incidence in the world grew by 20% in the last decade. Zinc, an essential trace element, is involved in many cellular processes. Concerning prostate cancer, Zn could inhibit the growth of tumor cells, by inducing cell cycle arrest or apoptosis. X-ray microfluorescence (μXRF) is an elemental analysis technique that allows mapping biologically important elements at a submillimeter scale, with high sensitivity and negligible damage to the sample. In this study, we investigated cell viability through colorimetric cytotoxicity assay (MTT) and the behavior of human prostate cell lines obtained from normal (RWPE-1) and tumorigenic (DU145) epithelium, in three-dimensional spheroid cultures after supplementation with ZnCl 2 for 24 and 48 hr using synchrotron μXRF. The measurements were performed at the Synchrotron Light National Laboratory (Campinas, Brazil). The results of μXRF showed that Zn intensity decreased in the DU145 tumor cells independent of supplementation or treatment time, and in normal cells, the intensity increased with supplementation and treatment time. The MTT assay results showed no significant differences, which indicates that the cell viability did not change. Our results indicate that μXRF can be used as an important tool to understand the mechanism of the loss of ability to capture zinc by prostate cancer cells.