Aging or acute loss of estrogens or androgens increases the levels of reactive oxygen species, activates nuclear factor-κB (NF-κB), and promotes the phosphorylation of p66(shc), a redox enzyme that amplifies mitochondrial reactive oxygen species generation and stimulates apoptosis. We report that in mesenchymal progenitor and osteoblastic cell models, H(2)O(2) activated a protein kinase C (PKC)β/p66(shc)/NF-κB signaling cascade and that p66(shc) was an essential mediator of the stimulating effects of H(2)O(2) on the apoptosis of osteoblastic cells as well as their ability to activate NF-κB. 17β-Estradiol (E(2)) or the nonaromatizable androgen dihydrotestosterone abrogated the effects of H(2)O(2) on p66(shc) and NF-κB activation by attenuating the phosphorylation of the redox-sensitive cytoplasmic kinase PKCβ. Additionally, both E(2) and dihydrotestosterone prevented H(2)O(2)-induced apoptosis by a mechanism that involved attenuation of p66(shc) resulting from decreased phosphorylation of PKCβ. Consistent with a kinase-mediated mechanism of sex steroid action, the effects of E(2) were reproduced by a polymeric form of estradiol that is not capable of stimulating the nuclear-initiated actions of ERα. These results demonstrate that p66(shc) is an essential mediator of the effects of oxidative stress on osteoblastic cell apoptosis, NF-κB activation, and cytokine production. The ability of either estrogen or androgen to attenuate the effects of oxidative stress on osteoblastic cell apoptosis, NF-κB activation, and cytokine production results from their common property to suppress PKCβ-induced p66(shc) phosphorylation via a mechanism that does not require stimulation of the nuclear-initiated actions of sex steroids.