Objective: Cytohesins are guanine nucleotide exchange factors involved in the PI3-K signalling pathway. In endothelial cells (EC) vascular endothelial growth factor (VEGF) is an important stimulus of PI3-K activation. It is unknown whether cytohesins are present in EC and play a role in VEGF dependent endothelial signalling and angiogenesis. We investigated the influence of cytohesins on VEGF dependent proliferation and PI3-K signalling in human microvascular (HMEC) and human umbilical endothelial cells (HUVEC). Methods: Cytohesin mRNA and protein expression were detected by RT-PCR, Western Blot and immunofluorescence staining. EC-proliferation was measured by MTT reduction. Results: On mRNA and protein levels, the presence of Cytohesin-1, -2 and -3 in HUVEC and HMEC was confirmed; expression of Cytohesin-2 was strongest. Addition of the pharmacological cytohesin-inhibitor SecinH3 (10mM) impaired the VEGF (50ng/ml) dependent proliferation considerably by 19±5% (p<0.05, n=12, HMEC). Moreover VEGF dependent activation of Akt was impaired after treatment with SecinH3 (15mM, p<0.05, n=6, HMEC). In contrast, VEGF induced phosphorylation of the MAP kinase ERK1/2 was still functional after cytohesin inhibition (n=6, HMEC), whereas the pharmacological inhibition of PI3-K prevents this (Wortmannin, 10nM, n=3). After stimulation with VEGF (50ng/ml, 10min) cell membrane recruitment and dimerisation of Cytohesin-2 increased. Inhibition of PI3-K (LY294006, 10mM) diminished this response. Interestingly Cytohesin-2 recruitment to the cell membrane was also impeded by pharmacological inhibition of tyrosine phosphatase SHP-2 (PtpI IV, 2mM, n=4, HMEC). SHP-2 could be activated by VEGF (p<0.05, n=7, HMEC). Knock out of this phosphatase prevented the VEGF dependent EC-proliferation (AS-ODN, p<0.05, n=12, HMEC). Conclusion: These results indicate that cytohesins, in particular cytohesin 2 are important for VEGF dependent PI3-K signalling and that cytohesin-2 may be regulated by SHP-2, which thus influences VEGF dependent EC-proliferation.