Tumor cells and their surrounding microenvironment produce a variety of factors that promote tumor growth and metastasis. We recently identified a nuclear factor, termed com1, that is up-regulated in human breast carcinoma cells on formation of experimental metastatic tumors and is assumed to act as a growth-promoting factor in breast cancer. 1,25-Dihydroxyvitamin D₃[1,25(OH)₂D₃] is a potent inhibitor of growth in breast cancer both in vitro and in vivo. We compared the growth-regulatory mechanisms of nontumorigenic and estrogen-dependent MCF-7 cells with those of the tumorigenic and tamoxifen-resistant subline MCF7/LCC2 in the presence of 1,25(OH)₂D₃. Proliferation of MCF7/LCC2 cells, which revealed constitutive com1 expression, was inhibited by 1,25(OH)₂D₃(10⁻⁷ m). This was strongly associated with cell cycle arrest in G₁ phase, consistent with accumulation of the hypophosphorylated form of the retinoblastoma protein as well as the induction of the cyclin-dependent kinase inhibitor p21. These cell cycle events were preceded by a transient up-regulation (5–8-fold) of com1 mRNA. Furthermore,clonal growth of the MCF7/LCC2 cells was also inhibited by 1,25(OH)₂D₃ (10⁻⁷m), and when the com1-negative MCF-7 cells were stably transfected with com1, the resulting MCF7/com1 cells showed a significant decrease in colony formation. These results seem to indicate that rather than promoting growth, com1 may participate in the regulatory pathway involved in cellular growth inhibition when recruited by inhibitory signals.