To study the role of la,25-dihydroxyvitamin D3 (calcitriol) in tumor promotion, we used JB6 C141.5a cells, a mouse epidermal cell model of tumor promotion. The phorbol ester 12-0-tetradecanoylphorbol-13-ace-tte (TPA) irreversibly induces anchorage-independent growth and tumorigenicity in these cells. Since we previously showed that calcitriol does not transform these cells but inhibits their proliferation, we hypothesized that calcitriol would inhibit TPA-induced transformation. Concurrent treatment of JB6 C141.5a cells with TPA and calcitriol revealed that calcitriol enhanced (1.7- to 10-fold, depending on dose) TPA-induced anchorage-independent growth without enhancing cell proliferation. Furthermore, a more than additive effect on osteopontin mRNA and protein levels was observed with concurrent drug treatment, which yielded a more highly phosphorylated form of osteopontin. These studies suggest coordinate regulation between the signaling pathways for calcitriol and TPA in JB6 C141.5a cells and further implicate expression of phosphorylated osteopontin in tumorigenesis. © 1993, American Association for Cancer Research. All rights reserved.