Generation of reactive oxygen species (ROS) by plasma membrane-localized NADPH oxidase (Nox 2) is a major mechanism of cell signaling associated with activation of the enzyme by a variety of agonists. With activation, the integral membrane flavocytochrome of Nox 2 transfers an electron from intracellular NADPH to extracellular O(2), generating superoxide anion (O(2)(*-)). The latter dismutes to H(2)O(2) which can diffuse through aquaporin channels in the plasma membrane to elicit an intracellular signaling response. O(2)(*-) also can initiate intracellular signaling by penetration of the cell membrane through anion channels (Cl(-) channel-3, ClC-3). Endosomes containing Nox2 and ClC-3 (called signaling endosomes) are composed of internalized plasma membrane and generate O(2)(*-) in the endosomal lumen to initiate signaling at intracellular sites. Thus, cellular signaling by Nox2 is dependent on the transmembrane flux of ROS. The role of this pathway has only recently been described and will require additional investigation to appreciate its physiological significance fully.