Abstract Human colon carcinoma (HT-29) cells were examined for their capacity to bind and respond to 1,25-dihydroxycholecalciferol [1,25-(OH) 2D 3]. These cells are known to differentiate and increase their population doubling time when galactose is substituted for glucose in their media. High-affinity and specific binding of 1,25-(OH) 2[ 3H]D 3 was observed in extracts of these cells grown in glucose. The binder sedimented in sucrose gradients and eluted from DEAE-cellulose columns in a manner indistinguishable from rabbit intestinal 1,25-(OH) 2D 3-receptor. Smaller amounts of this binder were seen in HT-29 cells grown in galactose. Both glucose-fed and galactose-fed cells exhibited a dose-dependent decrease in growth rate on exposure to 10 −12 to 10 −6 mol/L 1,25-(OH) 2D 3. Ultrastructural examination of galactose-fed and glucose + 1,25-(OH) 2D 3-treated cells showed enterocytic differentiation and features that were not distinguishable between these groups. Sucrase activity was higher in galactose-fed cells and did not change with 1,25-(OH) 2D 3 treatment. However, the lower sucrase activity in glucose-fed cells increased after exposure to 10 −8 mol/L 1,25-(OH) 2D 3. These results indicate receptor content and bioresponsivity to 1.25-(OH) 2D 3 in a human enterocytic cell line, suggesting that it will be a useful model for the study of the mechanisms of action of this sterol.