Fabry disease results from an X-linked mutation in the lysosomal alpha-galactosidase A (Gla) gene. Defective Gla results in multi-organ accumulation of neutral glycosphingolipids (GSLs), especially in the vascular endothelium, with the major GSL accumulated being globotriaosylceramide (Gb3). Excessive endothelial Gb3 accumulation is associated with increased thrombosis, atherogenesis and endothelial dysfunction. However, the mechanism(s) by which endothelial dysfunction occurs is unclear. The purpose of the present study was to further characterize the vasculopathy associated with a murine model of Fabry disease. Vascular reactivity was performed in vessels from wild-type (Gla(+/0)) and Gla-knockout (Gla(-/0)) mice. Conscious blood pressure and heart rate were measured in Gla(+/0) and Gla(-/0) mice by telemetry. The present study demonstrates that vascular smooth muscle (VSM) contractions to phenylephrine and serotonin, but not to U46619, were blunted in Gla(-/0) mice. Endothelium-dependent contraction and receptor-mediated endothelium-dependent relaxation to acetylcholine were significantly attenuated in vessels from Gla(-/0) mice. However, receptor-independent endothelium-dependent relaxation to the calcium ionophore ionomycin remained intact in vessels from Gla(-/0) mice. Furthermore, VSM reactivity was normal in aortas from Gla(-/0) mice in the absence of endothelium. These changes in vascular function were observed without changes in whole-animal blood pressure or heart rate. These results suggest that the vasculopathy associated with Fabry disease is localized to the endothelium, despite the accumulation of GSLs throughout the vasculature.