The present study was conducted on peripheral blood lympho-cytes of rainbow trout (Oncorhynchus mykiss) to assess the role of 5-hydroxytryptamine (5-HT; ‘serotonin’) in calcium signalling. 5-HT-induced increases in intracellular free calcium concentrations, [Ca2+]i, and its action was mediated by 5-HT receptor subtype 3 (5-HT3), but not by 5-HT receptor subtype 1A (5-HT1A) or subtype 2 (5-HT2) in these cells. In Ca2+-containing medium (1 mM CaCl2), 5-HT and 2-methyl-5-HT (5-HT3 receptor agonist) induced increases in [Ca2+]i, whereas in Ca2+-free medium (0 Ca2+, 1 mM EGTA), these two agents failed to evoke increases in [Ca2+]i in these cells, demonstrating that 5-HT mobilizes Ca2+ from the extracellular environment. Furthermore, 5-HT-induced increases in [Ca2+]i are not contributed to by the intracellular endoplasmic reticulum (ER) pool, as thapsigargin, an agent that recruits Ca2+ from ER stores, had additive effects on 5-HT-induced [Ca2+]i responses in fish peripheral lymphocytes. 5-HT-induced increases in [Ca2+]i were mediated by 5-HT3 receptors via gating the calcium through L-type, but not N-type, calcium channels in trout lymphocytes.