Murine JB6 epidermal cells can be irreversibly transformed into tumorigenic cells by the tumor promoter, 12–0etradecanoylphorbol-13-acetate. One feature of this transformation is induction of the synthesis and secretion of the phosphoprotein osteopontin (also called secreted phosphoprotein 1 and previously referred to as transformation-related phosphoprotein, 2ar, bone sialoprotein 1, or Mr, 44,000 bone phosphoprotein), an arginylglycylaspartic acid-containing cell adhesion glycoprotein the expression of which has been implicated in tumorigenesis and metastasis. Since lα,25-dihydroxyvitamin D3, calcitriol, also transforms JB6 cells and, in other cell types, regulates osteopontin synthesis, we hypothesized that calcitriol-mediated transformation of JB6 cells would also cause induction of osteopontin synthesis and secretion. Metabolic labeling with 32P04 of near confluent JB6 cells (clone 41.5a) treated with calcitriol (0.1–100 ng/ml) for up to 48 h revealed only a minimal production of osteopontin, which is the major phosphoprotein secreted by 12-O-tetradecanoylphorbol-13-acetate-treated cells. Similar treatment followed by labeling with [35S]ethionine showed a substantial dose-dependent increase in the synthesis and secretion of osteopontin. This induction was not associated with increased cell proliferation or with cell transformation, as assayed by anchorage-independent growth. Cal-citriol-treated cells were morphologically indistinguishable from control cells, while 12-O-tetradecanoy I phorbol-13-acetate-treated cells acquired a distinctive morphology. No induction of osteopontin was found with 25-hydroxyvitamin D3 or 24A,25-dihydroxyvitamin D3. These results show that calcitriol induces the synthesis and secretion of a nonphosphorylated form of osteopontin, in a cell type which normally makes little or none of this protein, and that the induction is not correlated with the tumorigenic transformation of these cells. © 1991, American Association for Cancer Research. All rights reserved.