Vascular sympathetic innervation is an important determinant of blood pressure and blood flow. The mechanisms that determine vascular sympathetic innervation are not well understood. Recent studies indicate that vascular endothelial cells (EC) express semaphorin 3A, a repulsive axon guidance cue. This suggests that EC would inhibit the growth of axons to blood vessels. The present study tests this hypothesis. RT-PCR and Western analyses confirmed that rat aortic vascular ECs expressed semaphorin 3A, as well as other class 3 semaphorins (sema 3s). To determine the effects of EC-derived sema 3s on sympathetic axons, axon outgrowth was assessed in cultures of neonatal sympathetic ganglia grown for 72 hours in the absence and presence of vascular EC. Nerve growth factor-induced axon growth in the presence of ECs was 50 ± 4% (p < 0.05) of growth in the absence of ECs. ECs did not inhibit axon growth in the presence of an antibody that neutralized the activity of sema 3s (p > 0.05). RT-PCR and western analyses also indicated that sema 3s was expressed in EC of intact arteries. To assess the function of sema 3s in arteries, sympathetic ganglia were grown in the presence of arteries for 72 hours, and the percent of axons that grew towards the artery determined. 44 ± 4% of axons grew towards neonatal carotid arteries. Neutralization of sema 3s or removal of EC increased the percent of axons that grew towards the artery (71 ± 8% and 72 ± 8%, respectively). These data indicate that vascular EC-derived sema 3s inhibit sympathetic axon growth and may thus be a determinant of vascular sympathetic innervation.