Mycotoxins are considered to be significant contaminants of food and animal feed. Zearalenone (ZEA) is a hepatotoxic mycotoxin with estrogenic and anabolic activity found in cereal grains worldwide. ZEA affects hematological and immunological parameters in humans and rodents. The compound can induce cell death, cause lipid peroxidation, inhibit protein and DNA synthesis, and exert genotoxic effects. ZEA may cause increased phagolysosomal fragility in the kidney. Our research showed that exposure of human embryonic kidney (HEK293) cells to ZEA (10 or 20μM) resulted in a concentration-dependent increase in DNA strand breaks measured with the comet assay. Damage was reduced in cells pretreated with NH4Cl, pepstatin A, or desipramine for 1h. Production of reactive oxygen species (ROS) was increased in cells exposed to ZEA, but DNA strand break induction could not be inhibited by the antioxidant hydroxytyrosol (HT). These results suggest that oxidative stress does not play a key role in DNA strand breaks induced by ZEA, that lysosomal injury precedes DNA strand breaks, and that the lysosome may be a primary target for ZEA in HEK293 cells.