Osteopontin (OPN) is a secreted, adhesive glycoprotein. Elevated expression of OPN in malignant and benign tumors is postulated to play a role in tumorigenesis. To determine whether OPN induction is required for tumor promotion, we used the in vitro JB6 model known to correlate with tumor promotion in vivo. The skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces irreversible transformation of JB6 C141.5a cells. Concomitantly, TPA markedly stimulates early and persistent OPN expression and secretion for at least 4 days (the time required for these cells to begin to acquire the transformed phenotype) and increases cells' adhesion to OPN. Here, we demonstrated that dexamethasone, a synthetic analog of glucocorticoid, known to inhibit tumor promotion in vivo, not only suppressed TPA-induced OPN mRNA expression and inhibited tumorigenic transformation of JB6 C141.5a cells (as previously shown in JB6 C122 and C141 cells), but also that the addition of OPN partially restored dexamethasone suppression of TPA-induced cell transformation. Therefore, we tested the hypothesis that OPN induction is required for tumor promoter-induced transformation of JB6 cells by examining (i) whether the addition of OPN will induce transformation, (ii) whether antisense OPN expression will inhibit TPA-induced transformation and (iii) if the latter experiment showed inhibition of TPA-induced transformation whether the addition of OPN will rescue this effect. Results indicated that the addition of purified OPN induced a dose-dependent transformation of JB6 cells, as assessed by anchorage-independent growth assay and that this induction was suppressed by antibody to OPN. Furthermore, antisense OPN expressing JB6 clones suppressed TPA-induced OPN synthesis and secretion and inhibited TPA-induced anchorage-independent growth, which was partially rescued by the addition of OPN. In conclusion, OPN induction is required and can be sufficient to induce in vitro cellular transformation of a preneoplastic murine JB6 cell line.