Application of aroma extract dilution analysis (AEDA) on organic extracts from Port wines barrel-aged over 40 years revealed 5 odor-active compounds corresponding to descriptors used to qualify the characteristic old wine aroma. One of the compounds, described as "nutty" and "spicy-like", and present in at least 9 dilutions above the others, was perceived as particularly important. The compound responsible for this flavor was identified as 3-hydroxy-4,5-dimethyl-2(5H)-furanone (sotolon). The levels ranged from 5 to 958 μg/L for wines between 1 and 60 years old. It was also observed that during oxidative aging the concentration of this compound increased with time according to a linear trend (r > 0.95). Although the presence of 2-ketobutyric acid was verified, the constant rate of formation of sotolon with aging and its high correlation with sugar derivates (HMF, furfural) suggests other mechanisms, different from those reported for other wines. The flavor threshold of sotolon was evaluated in Port wine at 19 μg/L. Sensorial tests provided valuable information concerning sotolon impact on Port wine aroma. Samples supplemented with this substance were consistently ranked as older. In view of these results it can be expected that sotolon plays a pre-eminent role in the characteristic old Port wine aroma.