The mitoinhibitory effect of fumonisin B1 (FB1) on the mitogenic response of epidermal growth factor (EGF) was investigated in primary hepatocyte cultures with respect to the alterations in the omega6 fatty acid metabolic pathway. Fatty acid analyses of hepatocytes showed that EGF treatment resulted in a significant decrease in the relative levels of 20:4omega6 (arachidonic acid) and an increase in 18:2omega6 (linoleic acid). Supplementation of the hepatocyte cultures with 20:4omega6 in the absence of EGF resulted in an increase in the total omega6 and omega6/omega3 fatty acid ratio. Addition of 20:5omega3 (eicosapentaenoic acid) resulted in an increase of the relative levels of the long chain omega3 fatty acids at the expense of the omega6 fatty acids. When 20:4omega6 and 20:5omega3 was added in the presence of EGF, the mitogenic response of EGF was increased and decreased respectively. When compared to the fatty acid profiles in the absence of EGF, the decreased mitogenic response coincided with a decrease of total omega6 fatty acids and total polyunsaturated fatty acids (PUFA). In addition, the saturated and mono-unsaturated fatty acids increased and the polyunsaturated/saturated (P/S) fatty acid ratio decreased which implied a more rigid membrane structure. Addition of prostaglandin E2 (PGE2) and prostaglandin E1 (PGE1) stimulated and inhibited the mitogenic response respectively. Ibuprofen, a known cyclooxygenase inhibitor, and FB1 inhibited the EGF-induced mitogenic response in a dose-dependent manner. The mitoinhibitory effect of FB1 on the EGF response was counteracted by the addition of PGE2. FB1 also disrupts the omega6 fatty acid metabolic pathway in primary hepatocytes, resulting in the accumulation of C18:2omega6 in phospatidylcholine and triacylglicerol. The disruption of the omega6 fatty acid metabolic pathway and/or prostaglandin synthesis is likely to be an important event in the mitoinhibitory effect of FB1 on growth factor responses.