Some antitumor immunomodulators, such as a linear beta-1,3-D-glucan from Alcaligenes faecalis var. myxogenes IFO 13140 (TAK), induce potent tumoricidal activity of polymorphonuclear leukocytes (PMNs). In the present study we investigated the role of calcium on the tumoricidal activity of PMNs induced by immunomodulators, especially TAK. The calcium chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) almost completely inhibited TAK-induced PMN cytotoxicity and this inhibition was restored by Ca2+ but not by Mg2+. In Ca2+- and Mg2+-free medium, PMN cytotoxicity induced by TAK was recovered by the addition of Ca2+ provided that Mg2+ was also present. By scopoletin assay, hydrogen peroxide released from PMNs by TAK was also observed in the presence of Ca2+ but not in its absence. The PMN cytotoxicities induced by the other immunomodulators, Propionibacterium acnes, Bacillus Calmette-Guérin, zymosan A, and Nocardia cell wall skeletons were also Ca2+ dependent, judging from studies with EGTA and measurement of hydrogen peroxide release in the presence and absence of Ca2+. The Ca2+ dependency of these PMN cytotoxicities suggests that Ca2+ influx is involved in the cytolytic process, but PMN cytotoxicity was not induced by simple addition of the calcium ionophore A23187. Like TAK, phorbol myristate acetate induced PMN cytotoxicity but this cytotoxicity was not Ca2+ dependent. The present report demonstrates the difference in Ca2+ dependency of these PMN cytotoxicities; i.e., extracellular calcium was required for immunomodulator-induced PMN cytotoxicity, but not for phorbol myristate acetate-induced PMN cytotoxicity. This suggests that the processes of induction of PMN cytotoxicity by the two types of activators are not identical.