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This study set out to elucidate the mechanism by which H2O2generation is regulated in cultured porcine thyroid cells. We monitored continuously the effects of the calcium ionophore A23187 and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on H2O2generation, using homovanillic acid and horseradish peroxidase. A23187 and TPA stimulated H2O2generation. A23187 increased cytoplasmic free calcium and TPA activated protein kinase C. Generation of H2O2is therefore regulated by cytoplasmic free calcium and protein kinase C. Exposure to A23187 or TPA augmented further the stimulation of H2O2generation by TPA or A23187 respectively. Thus A23187 and TPA, by increasing cytoplasmic free calcium and activating protein kinase C respectively, synergistically activate H2O2generation.Journal of Endocrinology(1989) 120,503–508