The formation of (E)-2-butenal in oils subjected to temperatures applied in frying as well as in fried foods was evaluated. For this purpose, a sensitive and accurate stable isotope dilution essay was developed and validated using as an internal standard (E)-2-butenal d6 synthesized in the lab. Heating of corn oil, sunflower oil, extra virgin and refined olive oil, ω-3 frying oil and palm oil at 170 °C for a total of 8 hours resulted in the formation of (E)-2-butenal at levels between 0.05 to 1 μg g− 1and the highest amounts were already formed after two hours of heating. The highest (E)-2-butenal formation was observed in the oil containing ω-3 unsaturated fatty acids indicating that (E)-2-butenal content is mainly affected by the fatty acid composition. No significant amount of free (E)-2-butenal was detected in fried foods purchased from fast food shops with the exception of one chicken sample and a pork brochette. Further experiments including several foods did not indicate any increase of free (E)-2-butenal content due to frying. It can be suggested that fried foods may not be a significant dietary source of free (E)-2-butenal for humans. However, these results in combination with the available literature indicated that investigation of the contamination of the indoors environment where frying is taking place could be more relevant than the presence of free (E)-2-butenal in oils and foods.