In the present study, we demonstrate that E2F is implicated in the regulation of the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-BPase) during cell division. The expression of this enzyme is induced during the G₁/S transition of the cell cycle. We identified and monitored the E2F-pocket protein complexes that bind to the E2F site of the F-type promoter during cell-cycle entry, and we analysed their contribution to the phosphatidylinositol 3-kinase (PI 3-kinase)-mediated regulation of the promoter. We found that the predominant E2F complex bound to the F-type promoter in unstimulated/quiescent cells contains E2F4, DP1 and p130 proteins. In serum-stimulated (S-phase) cells, the composition of the complex switched to E2F1/4, DP1 and p107, together with cyclin A and cyclin-dependent kinase 2. Treatment with the PI 3-kinase specific inhibitor LY 294002 prevented the formation of the S-phase complex, suggesting that activation of the PI 3-kinase pathway is essential for the formation of this complex. Further supporting this idea, we obtained results showing that treatment of cycling NIH 3T3 cells with either wortmannin or LY 294002 induces the accumulation of the transcriptionally repressive p130—E2F4—DP1 complex. Using the Rat-1 ER—E2F1 cell line where E2F1 activity can be conditionally induced, we demonstrated that E2F activity is involved in the in vivo transcriptional regulation of the F-type 6PF2K/Fru-2,6-BPase gene. Taken together, our results show that the F-type 6PF2K/Fru-2,6-BPase is a genuine E2F-regulated gene, and that its regulation by the PI 3-kinase pathway is at least partially mediated through the E2F transcription factor.