The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca^(2+) concentrations ([Ca^(2+)]i) in HA59T human hepatoma cells is unclear. This study explored whether m-3M3FBS elevated basal [Ca^(2+)]i levels in suspended cells by using fura-2 as a Ca^(2+)-sensitive fluorescent dye. M-3M3FBS at concentrations of 10-50 μM increased [Ca^(2+)]i in a concentration-dependent fashion. The Ca^(2+) signal was reduced partly by removing extracellular Ca^(2+). M-3M3FBS-induced Ca^(2+) influx was inhibited by nifedipine, econazole, SK&F96365, aristolochic acid, and GF109203X. In Ca^(2+)-free medium, 50 μM m-3M3FBS pretreatment inhibited the [Ca^(2+)]i rise induced by the endoplasmic reticulum Ca^(2+) pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin partly reduced m-3M3FBS-induced [Ca^(2+)]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca^(2+)]i rise. At concentrations between 10 and 40 μM m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca^(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data suggest that m-3M3FBS induced apoptosis in a concentration-dependent manner. M-3M3FBS also increased levels of reactive oxygen species. Together, in human hepatoma cells, m-3M3FBS induced a [Ca^(2+)]i rise by inducing phospholipase C-independent Ca^(2+) release from the endoplasmic reticulum and Ca^(2+) entry via protein kinase C-sensitive store-operated Ca^(2+) channels. M-3M3FBS induced cell death that might involve apoptosis via mitochondrial pathways.