We invented a simultaneous measuring instrument of fluorescence and chemiluminescence, realizing the analysis of chronological correlation between change in intracellular Ca2+ concentration ([Ca2+](i)) and superoxide generation. A human monocytic cell line, THP-1, differentiated to be neutrophil-like cells generated superoxide with increase in intracellular Ca2+ concentration when stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP) whereas PMA, phorbol ester-stimulated Superoxide response occurred without change in [Ca2+](i). The cells treated with TMB-8, an intracellular Ca2+ antagonist, generated superoxide rapidly as well as transiently with transient [Ca2+](i) elevation after stimulation with fMLP, whereas EGTA-treated cells generated superoxide slowly as well as persistently with transient [Ca2+](i) elevation after the stimulation. These results suggest that the rapid and transient contents of superoxide generation are specific for Ca-i(2+) influx from the extracellular domain. Verapamil, voltage-dependent Ca2+ channel blocker, dose-dependently inhibited fMLP-stimulated extracellular Ca2+ influx and superoxide generation without affecting PMA-stimulated superoxide generation. Other channel blockers tested, nifedipine and diltiazem, similarly inhibited these fFMLP-stimulated responses. Numerical analysis of the values of the response curves elucidated that TMB-8 or the channel blocker reveals or eliminates the same contents of superoxide generation by the antagonism of intracellular Ca2+ release or extracellular Ca2+ influx, respectively. Taking these results together, the characteristic extracellular Ca2+ influx essential for superoxide generation was first revealed by the simultaneous measurement of superoxide generation and change in [Ca2+](i). (c) 2006 Elsevier Inc. All rights reserved.