Background: Oleic acid is the principal fatty acid of olive oil composition and is reported to play a crucial role in its healthy aspects. However, the detailed mechanism of action is poorly understood. Aims: This study aims to elucidate the role of oleic acid in calcium signaling in rat thymocytes, in comparison to linoleic and linolenic acid. Methods: Fatty acids were applied to thymocytes isolated from wistar rats and loaded with Fura-2 to measure calcium signals. Results: The main results showed a concentration-dependent increase in [Ca2+]i induced by the 3 fatty acids. Raising the number of unsaturations resulted in greater increases. Two different pathways contributed to the increase induced by the polyunsaturated fatty acids: an IP3- independent release from the thapsigargin-sensitive stores and an extracellular calcium entry by econazole and nifedipine-insensitive channels. However, the OA-induced increases in [Ca2+]i seemed to be due mostly to the Ca2+ recruited from the intracellular stores. Conclusion: This study demonstrates that the fatty acids tested induce increases in [Ca2+]i in rat thymocytes, with differences in close relation to the degree of unsaturation. Such differences could be responsible for their different physiological action.