FLT1 is a cell surface VEGF receptor which is cleaved to release an N-terminal ectodomain which binds VEGF and PlGF and can antagonize the effects of VEGF in the extracellular milieu. To further evaluate FLT1 processing we expressed tagged FLT1 constructs in HEK293 and COS7 cells where we demonstrate, by deletion mapping, that the cleavage site is immediately adjacent to the transmembrane domain (TMD) between residues 759 and 763. Cleavage reciprocally regulates free VEGF in conditioned media and we show that the cleavage site is also transferable to another transmembrane receptor. A second cleavage event downstream of the ectodomain cleavage releases a cytosolic C-terminal FLT1 fragment and this intracellular cleavage of FLT1 is not catalyzed or regulated by the upstream ectodomain cleavage since abolition of the ectodomain cleavage has no impact on the downstream cleavage event. The downstream cleavage event is not susceptible to γ-secretase inhibitors and overexpression of presenilin 1, the catalytic subunit of γ-secretase did not change the downstream intracellular cleavage event. Furthermore, this cleavage did not occur via a previously published valine residue (767 V) in the TMD of FLT1, indicating the existence of another cleavage pathway. We tested the impact of the ectodomain cleavage on p44/42 MAP kinase activation and demonstrate that compared to wild type FLT1, cleavage resistant FLT1 constructs failed to stimulate p44/42 MAP kinase activation. Our results indicate that FLT1 ectodomain cleavage not only regulates the availability of free VEGF in the extracellular milieu but also regulates cellular signaling via the ERK kinase pathway.