The cytosolic proteins p47phox and p67phox, each containing two SH3 domains, are required for activation of the superoxide-producing phagocyte NADPH oxidase in a cell-free system with human neutrophil membrane and the small GTPase Rac. Here we focus on roles of proline-rich regions (PRRs) that reside in p47phox and p67phox. Deletion of the p47phox PRR, to which the C-terminal SH3 domain of p67phox binds, results in three-fold decreased activation of the enzyme in the cell-free system with the full-length p67phox, suggesting a modulatory role of the p47phox PRR. The modulation is likely mediated via the C-terminal region of p67phox, since the p47phox mutant protein fully activates the oxidase in combination with the N-terminus of p67phox. Neither deletion of the p67phox PRR nor substitutions for prolines in the region affects the ability to support superoxide production under the cell-free conditions, indicating that the PRR of p67phox has no primary function in the oxidase activation.