We have established two sublines derived from the insulin-secreting mouse pancreatic β-cell line MIN6, designated m9 and m14. m9 Cells exhibit glucose-induced insulin secretion in a concentration-dependent manner, whereas m14 cells respond poorly to glucose. In m14 cells, glucose consumption and lactate production are enhanced, and ATP production is largely through nonoxidative pathways. Moreover, lactate dehydrogenase activity is increased, and hexokinase replaces glucokinase as a glucose-phosphorylating enzyme. The ATP-sensitive K⁺channel activity and voltage-dependent calcium channel activity in m14 cells are reduced, and the resting membrane potential is significantly higher than in m9 cells. Thus, in contrast to m9, a model for β-cells with normal insulin response, m14 is a model for β-cells with impaired glucose-induced insulin secretion. By mRNA differential display of these sublines, we found 10 genes to be expressed at markedly different levels. These newly established MIN6 cell sublines should be useful tools in the analysis of the genetic and molecular basis of impaired glucose-induced insulin secretion.