Among the Shc proteins, p66shc is known to be related to oxidative stress responses and regulation of the production of reactive oxygen species (ROS). The present study was undertaken to investigate the role of p66shc on endothelial nitric oxide synthase (eNOS) activity in the mouse embryonic fibroblasts (MEFs). When wild type (WT) or p66shc (⁣/⁣) MEFs were transfected with full length of eNOS cDNA, the expression and activity of eNOS protein were higher in the p66shc (⁣/⁣) MEFs. These phenomena were reversed by reconstitution of p66shc cDNA transfection in the p66shc (⁣/⁣) MEFs. The basal superoxide production in the p66shc (⁣/⁣) MEFs was not significantly different from that of WT of MEFs. However, superoxide production induced by NADPH in the p66shc (⁣/⁣) MEF was lesser than that in WT MEFs. When compared with WT MEFs, cell lysate of p66shc (⁣/⁣) MEFs showed significantly increased H-ras activity without change of endogenous H-ras expression. Our findings suggest the pivotal role of p66shc adaptor protein played in inhibition of endothelial nitric oxide production via modulation of the expression and/or activity of eNOS protein.