At the level of the whole animal, the toxic effects of the mycotoxin deoxynivalenol (DON) range from causing diarrhoea, vomiting, gastro-intestinal inflammation to necrosis of several tissues. It also affects the immune system and leads to kidney lesions. Although DON has been tested in different human and animal cell lines for its cytotoxicity, these tests might be limited due to the disadvantages of cell lines (e.g. immortalization, tumour derivation, longtime cultivation) and do not necessarily reflect the response of normal cells. In order to overcome this problem and to be closer to the human situation, we studied the effect of DON in human kidney epithelial cells (renal proximal tubule epithelial cells, RPTEC) and human lung fibroblasts (normal human lung fibroblast, NHLF) in primary culture. Cell viability, apoptotic and necrotic cell death, collagens I, III and IV as well as fibronectin secretion were determined. It could be demonstrated that DON has a distinct cytotoxic effect on human primary cells. A reduction in viability can be observed in both cell types, with fibroblasts reacting more sensitive. Furthermore, DON caused mainly necrotic cell death in kidney cells whereas mainly apoptotic cell death in fibroblasts. DON had no effect on collagen secretion in RPTEC cells. Collagen secretion was partially decreased in NHLF. In both cells, fibronectin secretion was reduced after 5 days of exposure. We also studied the metabolism and the cellular uptake of DON using LC-MS/MS. DON was neither metabolized by proximal tubule cells nor by fibroblasts. DON is incorporated into the cells whereas the intracellular amount of DON in kidney cells is higher than in fibroblasts. No accumulation of DON occurred in the cells.