Effects of insulin on key steps of carbohydrate metabolism were investigated in cultured HT29 colon cancer cells by two different approaches, i.e. incubation of the cells either in the absence or in the presence of glucose in the medium. In glucose-deprived cells, insulin decreased glycogen breakdown, but did not affect polysaccharide levels when glucose was present. Glycogen synthase became activated after insulin treatment in both conditions, even though the activation was more evident when glucose was omitted. No effect on glycogen phosphorylase activity was evident under our experimental conditions. In cells incubated with glucose, the hormone stimulated in a dose-dependent manner the rates of glucose uptake and lactate release. Concomitantly with the increase in glycolytic rate, insulin caused a strong increase in fructose 2,6-bisphosphate. This effect was not observed in the absence of glucose. It is concluded that the carbohydrate metabolism of cultured HT29 cells responds to insulin, making this biological model suitable for investigations in vitro on the mechanism of insulin action.