Proliferating and tumour cells express the glycolytic isoenzyme, pyruvate kinase type M2 (M2-PK), which occurs in a highly active tetrameric form and in a dimeric form with low affinity for phosphoenolpyruvate. The switch between the two forms regulates glycolytic phosphometabolite pools and the interaction between glycolysis and glutaminolysis. In the present study, we show the effects of oncoprotein E7 of the human papilloma virus (HPV)-16 (E7)-transformation on two NIH 3T3 cell strains with different metabolic characteristics. E7-transformation of the high glycolytic NIH 3T3 cell strain led to a shift of M2-PK to the dimeric form and, in consequence, to a decrease in the cellular pyruvate kinase mass-action ratio, the glycolytic flux rate and the (ATP+GTP)/(UTP+CTP) ratio, as well as to an increase in fructose 1,6-bisphosphate (FBP) levels, glutamine consumption and cell proliferation. The low glycolytic NIH 3T3 cell strain is characterized by high pyruvate and glutamine consumption rates and by an intrinsically large amount of the dimeric form of M2-PK, which is correlated with high FBP levels, a low (ATP+GTP)/(CTP+UTP) ratio and a high proliferation rate. E7-transformation of this cell strain led to an alteration in the glycolytic-enzyme complex that correlates with an increase in pyruvate and glutamine consumption and a slight increase in the flow of glucose to lactate. The association of phosphoglyceromutase within the glycolytic-enzyme complex led to an increase of glucose and serine consumption and a disruption of the linkage between glucose consumption and glutaminolysis. In both NIH 3T3 cell lines, transformation increased glutaminolysis and the positive correlation between alanine and lactate production.