The effect of 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a Ca2+ pump inhibitor, on superoxide anion (O2-) production was examined with a special reference to Ca2+ in HL-60 cells differentiated by dibutyryl cAMP, and compared with the effect of N-formyl-Met-Leu-Phe (fMLP) and phorbol 12-myristate 13-acetate (PMA). tBuBHQ caused O2- production and Ca2+ mobilization, but not phosphoinositide hydrolysis. fMLP caused O2- production, Ca2+ mobilization and phosphoinositide hydrolysis. PMA caused O2- production without affecting Ca2+ mobilization and phosphoinositide hydrolysis. EGTA and O,O'-bis(2-aminophenyl)ethyleneglycol- N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM), an intracellular Ca2+ chelator, inhibited O2- production induced by fMLP, but not by tBuBHQ. Thapsigargin, another Ca2+ pump inhibitor, had a weak ability to produce O2-. fMLP, but not tBuBHQ, caused BAPTA/AM-sensitive activation of phospholipase A2 and D. tBuBHQ caused O2- production by interacting with phosphatidylcholine in a cell-free system. The results suggest that tBuBHQ causes O2- production independent of Ca2+, and Ca2+ might be a cofactor in the activation of phospholipase A2 and D upstream in fMLP-induced O2- production.