To investigate the underlying mechanisms of C18 fatty acids (stearic acid, oleic acid, linoleic acid and α-linolenic acid) on mast cells, we measured the effect of C18 fatty acids on intracellular Ca2+ mobilization and histamine release in RBL-2H3 mast cells. Stearic acid rapidly increased initial peak of intracellular Ca2+ mobilization, whereas linoleic acid and α-linolenic acid gradually increased this mobilization. In the absence of extracellular Ca2+, stearic acid (100 μM) did not cause any increase of intracellular Ca2+ mobilization. Both linoleic acid and α-linolenic acid increased intracellular Ca2+ mobilization, but the increase was smaller than that in the presence of extracellular Ca2+. These results suggest that C18 fatty acid-induced intracellular Ca2+ mobilization is mainly dependent on extracellular Ca2+ influx. Verapamil dose-dependently inhibited stearic acid-induced intracellular Ca2+ mobilization, but did not affect both linoleic acid- and α-linolenic acid-induced intracellular Ca2+ mobilization. These data suggest that the underlying mechanism of stearic acid, linoleic acid and α-linolenic acid on intracellular Ca2+ mobilization may differ. Linoleic acid and α-linolenic acid significantly increased histamine release. Linoleic acid (C18:2: ω-6)-induced intracellular Ca2+ mobilization and histamine release were more prominent than α-linolenic acid (C18:3: ω-3). These data support the view that the intake of more α-linolenic acid than linoleic acid is useful in preventing inflammation.