AbstractIn order to study the effect of glucose on the differentiation of cultured human colon cancer cells, a subpopulation of HT‐29 cells was selected for its capacity to grow in the total absence of sugar. These cells (GIc−cells) exhibit, after confluency, an enterocytic differentiation, in contrast to cells grown with glucose (Glc+cells), which always remain undifferentiated. The differentiation is characterized by a polarization of the cell layer with apical brush borders and tight junctions, and by the presence of sucrase‐isomaltase. The differentiation of Glc−cells is reversible: the addition of glucose to postcon‐fluent cultures of Glc−cells results in an inhibiting effect on the expression of sucrase‐isomaltase; switching growing cultures of Glc−cells to the Glc+medium for several passages results in a progressive reversion to the undifferentiated state, which is completed after seven passages. The dedifferentiation process is associated with a parallel, passage‐related, increase in the rates of glucose consumption and lactic acid production, and decreases of intracellular glycogen content, which return to the values of the undifferentiated original Glc+cells. The values of these metabolic parameters are correlated, at each passage, with the degree of dedifferentiation of the cells. When these dedifferentiated cells, after having been cultured in Glc+medium for 20 passages, are switched back to the Glc−medium, they readily grow without mortality, and reexpress the same enterocytic differentiation as the parent Glc−cells. These results show that the capacity of this subpopulation to grow and differentiate in the absence of sugar is a stable characteristic. They further suggest that glucose metabolism interferes with the program of different