1,25-Dihydroxyvitamin D 3 (1,25-(OH) 2D 3) greatly enhances sodium butyrate (NaB)-induced enterocyte differentiation of HT-29 human colonic carcinoma cells while 1,25-(OH) 2D 3 alone induces growth restriction without associated differentiation. In the present study, the efficacies of various analogs of 1,25-(OH) 2D 3 to enhance NaB-induced HT-29 differentiation and to prolong the reversal of the differentiated phenotype under NaB-free growth conditions were subsequently examined. Extent of HT-29 differentiation was assessed by measurement of alkaline phosphatase (AP) activity, appearance of mucin-producing cells, changes in morphological characteristics, and expression of differentiation-associated cytokeratin proteins. Among active analogs of 1,25-(OH) 2D 3, 26,26,26,27,27,27-hexafluoro-1,25-(OH) 2D 3 (F 6-1,25-(OH) 2D 3), 24,24-difluoro-24-homo-1,5-(OH) 2D 3, and 26,27-dimethyl-1,25-(OH) 2D 3 were 100-, 10-, and 5-fold, respectively, more effective than 1,25-(OH) 2D 3 in enhancing NaB-induced mucin production. Combined use of NaB and F 6-1,25-(OH) 2D 3 (10 −9 m) also induced HT-29 cells to form highly differentiated goblet-like enterocytes, and increased both cellular AP enzymatic activity and tissuetype cytokeratin content. This differentiated state was qualitatively more advanced than that achieved by a combination of NaB and 10 −7 m 1,25-(OH) 2D 3. NaB-mediated HT-29 differentiation (in short-term inductions) was found to be reversible following a return to NaB-free medium. HT-29 cells differentiated by combined use of NaB and 1,25-(OH) 2D 3 or its analogs exhibited a significant prolonged reversal time relative to cells differentiated with NaB alone. The most prominent effect was achieved using cells differentiated with NaB and 10 −9 mF 6-1,25-(OH) 2D 3 which exhibited a 7-fold prolonged reversal time over colonocytes differentiated by NaB alone. Our data suggest that a combined use of NaB and 1,25-(OH) 2D 3 or its derivatives may provide a convenient in vitro model system to probe molecular events associated with steroid-target tissue interactions in a differentiating cell system as commonly occurs in vivo. Such an analysis might lend itself to design of a rational combination differentiation-based therapy for the clinical management of colon cancer.