AIM: To evaluate the effects of gliadin on the oxidative environment in the "in vivo-like" model of a three-dimensional cell culture system. METHODS: LoVo cell line (intestinal adenocarcinoma) multicellular spheroids were treated with digested gliadin (with albumin used as a control). Spheroid volumes, cell viability and morphology, lactate dehydrogenase (LDH) release, content of reduced glutathione (GSH) and activity of GSH-related enzymes were examined. The data were statistically analyzed using the Student's t-test. was considered statistically significant. RESULTS: Gliadin reduced cell viability (from 20% to 60%) and led to morphological alterations characterized by apoptotic findings and cytoskeletal injuries. LDH activity increased. The content of GSH reduced (-20% vs controls), and activity of GSH-related enzymes was significantly inhibited. CONCLUSION: Gliadin treatment induces an imbalance in the antioxidative mechanism of cells cultured by the three-dimensional technique. This alteration may explain the cell damage directly caused by gliadin and the subsequent morphological abnormalities.