The last common ancestor of hagfish and gnathostomes was also the last common ancestor of all extant vertebrates that lived some time more than 500 million years ago. Features that are shared between hagfish and gnathostomes can be inferred to have already been present in this ancestral vertebrate. We recently reported that hagfish endothelium displays phenotypic heterogeneity in ultrastructure, lectin binding, and mechanisms of leukocyte adhesion. Thus, phenotypic cell heterogeneity evolved as an early feature of the endothelium. In the present study, we wanted to extend these observations by determining whether hagfish endothelium plays a role in mediating vasomotor tone. Response of mesenteric and skeletal muscle arteries to a variety of mediators was assayed by videomicroscopy. Phenylephrine and acetylcholine induced vasoconstriction of mesenteric and skeletal muscle arteries. Bradykinin (BK) and ADP promoted vasorelaxation in precontracted mesenteric arteries but not those from skeletal muscle. BK- and ADP-mediated vasorelaxation of the mesenteric artery was abrogated by mechanical denudation of the endothelium but was unaffected by N(G)-nitro-L-arginine methyl ester. Indomethacin significantly inhibited the vasodilatory response to ADP but not BK. The nitric oxide donor sodium nitroprusside resulted in endothelium-independent relaxation of both mesenteric and skeletal muscle arteries. Together, these data suggest that site-specific endothelium-dependent vasorelaxation is an evolutionarily conserved property of this cell lineage.