Syndecan-4 is one of the principal heparan sulfate-carrying proteins on the cell surface. Unlike other members of syndecan family, syndecan-4 mediates phosphatidylinositol 4,5-bisphosphate 2 (PIP(2))-dependent PKC-alpha activation, and overexpression of syndecan-4 in vitro results in enhanced FGF2 signaling. The present study was designed to test the functional effect of increased syndecan-4 expression in endothelial cells in transgenic mice. Several transgenic mice lines expressing syndecan-4 cDNA under control of human endothelial nitric oxide (NO) synthase (eNOS) promoter were generated. Exogenous syndecan-4 was mainly expressed in the heart, brain, and lungs. In particular, the heart demonstrated the greatest increase in the ratio of transgenic-to-native syndecan-4 gene expression. Vessels from the eNOS-syndecan-4 mice demonstrated more pronounced vasodilation to FGF2 but not to VEGF-A(165), sodium nitroprusside, and A 23187 compared with wild-type mice. To elucidate the mechanism of this effect, we measured NO release from primary cardiac endothelial cells isolated from transgenic or wild-type adult mice. Cells from the eNOS-syndecan-4 transgenic mice had a significant increase in FGF2- and VEGF-A(165)-induced NO release compared with endothelial cells from the wild-type mice. However, the absolute magnitude of this increase was higher for FGF2 than VEGF-A(165). In conclusion, enhanced syndecan-4 expression in mouse cardiac endothelial cells results in preferential augmentation of FGF2 but not VEGF-A(165)-induced NO release.