This paper deals with the mechanisms of activation of NADPH oxidase investigated using EBV-transformed human B lymphoblastoid cell lines (B cells) from normal subjects and from patients affected by X-linked chronic granulomatous disease (CGD). The results reported are as follows. 1) In normal B cells, the NADPH oxidase components p67 phox, p40 phox, p22 phox, and gp91 phox were less expressed than in polymorphonuclear neutrophils. 2) In normal B cells stimulated with PMA, p47 phox, p67 phox, and p40 phox translocated to the membranes as occurs in polymorphonuclear neutrophils. 3) In CGD, B cells expressing p22 phox in the absence of gp91 phox, p47 phox, p67 phox, and p40 phox did not translocate to the membranes after stimulation with PMA. 4) In PMA-stimulated B cells from an X91+ CGD patient in which p22 phox was normally expressed and gp91 phox was present but lacked five amino acids, translocation of p47 phox to the membranes was unaffected, but p67 phox and p40 phox were poorly translocated, and the production of O 2− was greatly reduced with respect to that by normal B cells. Taken together, these findings indicate that 1) a low expression of some NADPH oxidase components may represent the molecular basis of the low production of O 2− in B lymphocytes; 2) the cytosolic components of NADPH oxidase cannot bind to p22 phox on the membranes in the absence of gp91 phox; 3) p47 phox can translocate to the membranes independently of p67 phox and p40 phox; and 4) gp91 phox may have a role in mediating and/or stabilizing the binding of p67 phox and p40 phox to the membranes of activated cells.