The signal transduction pathways of transforming growth factor ß1 (TGF-ß1) and 1,25-dihydroxyvitamin D₃ [1,25(OH) ₂D₃ ], the major active biological metabolite of vitamin D₃ , have been shown to interact in several cell types. 1,25(OH) ₂D₃ inhibits the growth of many cancer cells, including Caco-2 cells, a human colon cancer-derived cell line. Whereas the growth of normal colonic epithelial cells is inhibited by TGF-ß1, most human colon cancer-derived cells, including Caco-2 and SW480 cells, are resistant to TGF-ß1. The mechanisms underlying the antiproliferative actions of 1,25(OH) ₂D₃ and the resistance to TGF-ß growth inhibition, however, remain largely unknown. In the present studies, we observed that 1,25(OH) ₂D₃ sensitized Caco-2 cells, as well as SW480 cells, to TGF-ß1 growth inhibitory effects. Compared with 1,25(OH) ₂D₃ alone, the combination of 1,25(OH) ₂D₃ (100 nM) and TGF-ß1 (2 ng/ml) caused a significant reduction in Caco-2 and SW480 cell numbers. In addition, we observed that the amount of active TGF-ß1 was increased (~4-fold) by this secosteroid in conditioned media from Caco-2 cells. 1,25(OH) ₂D₃ increased the expression of insulin-like growth factor II receptors (IGF-IIR) in Caco-2 cells, which facilitated activation of latent TGF-ß1. 1,25(OH) ₂D₃ was found to activate TGF-ß signaling in Caco-2 cells. Furthermore, by using neutralizing antibodies to human TGF-ß1, we demonstrated that this cytokine contributes to the secosteroid-induced inhibition of Caco-2 cell growth. 1,25(OH) ₂D₃ was also found to enhance the type I TGF-ß receptor mRNA and protein abundance in Caco-2 cells. Whereas the 1,25(OH) ₂D₃ -induced sensitization of Caco-2 cells to TGF-ß1 was IGF-IIR-independent, the type I TGF-ß receptor expression was required for this sensitization. Taken together, these studies have demonstrated that 1,25(OH) ₂D₃ treatment of Caco-2 cells resulted in activation of latent TGF-ß1, which was facilitated by the enhanced expression of IGF-IIR by this secosteroid. In addition, 1,25(OH) ₂D₃ sensitized Caco-2 cells to growth inhibitory effects of TGF-ß1, which also contributed to the inhibition of Caco-2 cell growth by this secosteroid.