Objective— Reactive oxygen species (ROS) that act as signaling molecules in vascular smooth muscle cells (VSMC) and contribute to growth, hypertrophy, and migration in atherogenesis are produced by multi-subunit NAD(P)H oxidases. Nox1 and Nox4, two homologues to the phagocytic NAD(P)H subunit gp91 ^phox , both generate ROS in VSMC but differ in their response to growth factors. We hypothesize that the opposing functions of Nox1 and Nox4 are reflected in their differential subcellular locations. Methods and Results— We used immunofluorescence to visualize the NAD(P)H subunits Nox1, Nox4, and p22 ^phox in cultured rat and human VSMC. Optical sectioning using confocal microscopy showed that Nox1 is co-localized with caveolin in punctate patches on the surface and along the cellular margins, whereas Nox4 is co-localized with vinculin in focal adhesions. These immunocytochemical distributions are supported by membrane fractionation experiments. Interestingly, p22 ^phox , a membrane subunit that interacts with the Nox proteins, is found in surface labeling and in focal adhesions in patterns similar to Nox1 and Nox4, respectively. Conclusions— The differential roles of Nox1 and Nox4 in VSMC may be correlated with their differential compartmentalization in specific signaling domains in the membrane and focal adhesions.