Hepatocyte-like mhAT3F cells have been derived from the hepatoma of a transgenic mouse expressing the SV40 large T antigen under the control of the antithrombin III gene regulatory region (Antoine, B., Levrat, F., Vallet, V., Berbar, T., Cartier, N., Dubois, N., Briand, P., and Kahn, A. (1992) Gene expression in hepatocyte-like lines established by targeted carcinogenesis in transgenic mice. Exp. Cell. Res. 200, 175-185; F. Levrat et al., unpublished results). In these cells, the L-PK gene is transcriptionally activated by glucose, as it is in vivo and in cultured hepatocytes. However, in contrast to the L-PK gene regulation in the liver and isolated hepatocytes, the glucose responsiveness does not require insulin and is not blocked by cyclic AMP. In mhAT3F cells, the insensitivity to insulin might be due to the replacement of insulin-dependent glucokinase by insulin-independent hexokinases able to phosphorylate glucose in the absence of the hormone. The glucose-dependent activation of the L-PK gene is delayed, requires ongoing protein synthesis, and is mediated by the same glucose response element as in vivo and in isolated hepatocytes. These results suggest that the glucose-dependent signaling pathway responsible for the transcriptional activation of glycolytic and lipogenic genes requires glucose phosphorylation, a phenomenon that is insulin-dependent in the liver but insulin-independent in cultured hepatoma cells. Nevertheless, the action of glucose 6-phosphate is most likely indirect.