Oleandrin, derived from the leaves of Nerium oleander, has been shown to possess anti-inflammatory and tumor cell growth-inhibitory effects. Here, we provide evidence that oleandrin could possess anti-tumor promoting effects. We determined the effect of topical application of oleandrin to CD-1 mice against l2-O-tetradecanoylphorbol-13-acetate (TPA), a widely studied skin tumor promoter, -induced conventional and novel markers of skin tumor promotion. Topical application of oleandrin (2 mg per mouse) 30 min before TPA (3.2 nmol per mouse) application onto the skin afforded significant inhibition, in a time-dependent manner, against TPA-mediated increase in cutaneous edema and hyperplasia, epidermal ornithine decarboxylase (ODC) activity and ODC and cyclooxgenase-2 (COX-2) protein expression. In search for novel markers of skin tumor promotion, we found that TPA application to mouse skin resulted, as an early event, in an increased expression of phosphatidyinositol 3-kinase (PI3K), phosphorylation of Akt at threonine308 and activation of nuclear factor kappa B (NF-kappaB). Topical application of oleandrin before TPA application to mouse skin resulted in significant reduction in TPA-induced expression of PI3K and phosphorylation of Akt, and inhibition of NF-kappaB activation. NF-kappaB is a eukaryotic transcription factor that is critically involved in regulating the expression of specific genes that participate in inflammation, apoptosis and cell proliferation. Employing Western blot analysis, we found that oleandrin application to mouse skin resulted in inhibition of TPA-induced activation of NF-kappaB, IKKalpha and phosphorylation and degradation of IkappaBalpha. Our data suggest that oleandrin could be a useful anti-tumor promoting agent because it inhibits several biomarkers of TPA-induced tumor promotion in an in vivo animal model. One might envision the use of chemopreventive agents such as oleandrin in an emollient or patch for chemoprevention or treatment of skin cancer.