Pii: 0006-291x(89)91357-0

a calcium ionophore, a phorbol ester, was studied. Upon stimulation with arachidonic acid or 7-hexachlorocyclohexane, Me$O-differentiated HL-60 cells generated superoxide (0~~) at higher rates than dbcAMP-differentiated cells. Undifferentiated cells generated 02only at low rates upon stimulation with the above agents. In cell-freesystems, NADPH oxidase activity was reconstituted by combining membranes of undifferentiated or dbcAMPor MegjO-differentiated HL-60 cells, cytosol of Me$O-differentiated cells and arachidonic acid. This basal 0 formation was enhanced several-fold by guanosine 5'-O-(3-thiotriphosphate) P vator of guanine nucleotide-binding proteins. GTP[$]), a potent actiIn contrast, cytosol of dbcAMPdifferentiated cells reconstituted 02formation only in the presence of GTP[+], and cytosol of undifferentiated cells was inactive. Submaximally stimulatory amounts of cytosolic protein of Me$Oand dbcAMP-differentiated cells synergistically stimulated 0 absence of GTP[+]. We conclude z formation in the presence but not in the hat differentiations of HL-60 cells with Me2S0 and dbcAMP are not equivalent with respect to activation of NADPH oxidase and that two cytosolic activation factors are involved in the regulation of this effector system.