The C-24 oxidation pathway plays a major role in the degradation of vitamin D metabolites in kidney and other target tissues. The aim of the present study was to establish an intestinal cell culture system to study the mechanisms regulating the vitamin D catabolic pathway. 25-Hydroxyvitamin D₃-24- hydroxylase (24-hydroxylase), the first enzyme in the catabolic sequence, was examined in Caco-2 cells, a human colon adenocarcinoma cell line which exhibits differentiated functions of absorbing intestinal epithelial cells. While untreated Caco-2 cells did not exhibit 24-hydroxylase activity, significant catabolic activity was induced by prior treatment of cell monolayers with 1,25-dihydroxyvitamin D3(1,25-(OH)₂D₃). Induced 24-hydroxylase activity was not inhibited by either 1,2-dianilinoethane or EDTA in the reaction mixture. 24-Hydroxylation of 25-hydroxyvitamin D₃ (₂₅OHD₃) and 1,25-(OH)₂D₃ was detected 6 h after treatment of cells with 10^-8 M 1,25-(OH)₂D₃, peaked at 16 h, and decreased thereafter. Treatment of cells with 10^-7 M 1,25- (OH)₂D₃ elicited a maximal 24-hydroxylase response. Comparable time courses of induction by 1,25-(OH)₂D₃ and 1,25- (OH)₂D₃-dose response curves were observed in cultured human skin fibroblasts and Caco-2 cells. 25OHD3 was not as good an inducer of the vitamin D catabolic pathway in Caco-2 cells as 1,25-(OH)₂D₃. Induction of 24-hydroxylase activity by 1,25- (OH)₂D₃ was inhibited by pretreatment of Caco-2 cells with either actinomycin D, a-amanitin, or cycloheximide suggesting that mRNA and protein synthesis are required for induction. The present study demonstrates that l,25-(OH)₂D₃-treated Caco-2 cells express the vitamin D catabolic pathway and, therefore, constitute a useful in vitro model to study the mechanism of induction by 1,25-(OH)₂D₃. (Endocrinology126: 2868– 2875, 1990)