Mitogen-regulated protein3 (MRP3) is a bFGF-regulated gene containing the basic-fibroblast growth factor response element (FRE) in its proximal promoter. The unique FRE is transcriptionally active in a TK fusion promoter and to respond to bFGF. The FRE transcriptional element is also present in the promoters of many bFGF-regulated genes like mmp-3, mmp-2, mmp-1, and mmp-9. The FRE binds to nuclear factors derived from NIH 3T3 cells (mouse), CHO cells (hamster), HeLa cells (human), and from tissues like placenta (day 11) and fetus, regions where mrp3 is highly expressed. This [Difference]34kDa nuclear factor, named the Fibroblast growth factor response element binding protein (FREBP) is proposed to be the transcription factor regulating FRE-containing genes. The FREBP was shown to be an anchorage dependent protein with its activity declining over a period of 24h in suspension cultures of HeLa cells. Studies were conducted using inhibitors for the enzymes Ras, MEK kinase, Src kinase, and PI-3 kinase to investigate the involvement of extracellular regulated pathways like MAP kinase, PI-3 kinase, and FAK pathway in regulating the FREBP activity. The results showed that the MEK inhibitor PD 98059 inhibits FREBP activity in cells that were adherent to the ECM (p<0.001). This result suggested that MEK might be involved in regulating the FREBP activity in monolayer HeLa cells. The inhibitors for the other molecules, like PP2 (Src kinase), Ly294002 (PI-3 kinase), and Ftase inhibitor (Ras), did not significantly reduce FREBP activity in monolayer cultures. To know whether MEK activity regulates FREBP activity in an anchorage dependent manner, the following studies were undertaken. First, activity of MEK established in suspended HeLa cells over a 24h time course. Second, effect of inhibiting MEK activity over a 24h time course determined. Results showed that MEK activity decreases more slowly than the FREBP activity in suspended HeLa cells (Half-life: FREBP: [Difference]2h, MEK: [Difference]18h). Thus, MEK does not inhibit FREBP activity in suspension cultures, and also inhibiting MEK did not have an effect on the FREBP activity in suspended cells. Based on our studies, we propose that MEK could be involved in regulating FREBP in Monolayer cells but not in suspended HeLa cells.