Macrophages produce a large volume of reactive oxygen species (ROS) through respiratory burst. However, the influence of inducible nitric oxide synthase (Inos) activation on ROS production remains unclear. In the present investigation, the kinetic generation of ROS in RAW264.7 murine macrophages was monitored by chemiluminescence. Phorbol 12- myristate 13-acetate (PMA) induces a robust chemiluminescence in RAW264.7 cells, suggesting protein kinase C (PKC)-related assembly and activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). The effects of Inos induction on ROS production were examined. Induction of In OS expression in RAW264.7 cells with lipopolysaccharide (LPS; 1 mg/ml) causes a significant increase in PMA-induced chemiluminescence, which could be enhanced by the NOS substrate, L-arginine (L-Arg), and could be abolished by the NOS inhibitor, L-NG-nitroarginine (L-NNA). Further experiments reveal that induction of Inos expression enhances the PMA-stimulated phosphorylation of the p47phox subunit of NOX, and promotes the relocalization of cytosolic p47phox and p67phox subunits to the membrane. Inhibition of PKCz by its myristoylated pseudosubstrate significantly decreased the PMA-stimulated phosphorylation of the p47phox in LPS-pretreated cells, suggesting that PKCz is involved in the Inos-dependent assembly and activation of NOX. Taken together, the present investigation suggests that the induction of Inos up-regulates the PMA-induced assembly of NOX and leads to the enhanced production of ROS via a PKCz-dependent mechanism.