Long-term behavior of rapeseed oil in waste activated bleaching earth (ABE) and the effect of this oil on riboflavin production in the culture of Ashbya gossypii were investigated. Waste ABE with 40% (w/w) rapeseed oil was stored for 80 d, and the extent of oxidation of rapeseed oil was measured by several analytical methods to determine the chemical properties of the oil at different stages of the oil deterioration process: peroxide value, acid value, concentrations of organic acids, acetaldehyde and unsaturated fatty acid, and content of polymerized triglycerides. Peroxide value, acid value, and concentrations of organic acids and acetaldehyde did not affect riboflavin production. However, the content of polymerized triglycerides markedly increased the viscosity of rapeseed oil and was the main reason for the exponential decrease in riboflavin production. A good correlation between the polymerized triglyceride content or viscosity and riboflavin production in the culture of A. gossypii using rapeseed oil as the sole carbon source was found.