UNLABELLED: BACKGROUND. There has been a significant amount of research on the effects of nicotine on vascular biology; however, little is known about the effects of cotinine, the metabolic product of nicotine. This study used a novel vascular perfusion system to study the effects of nicotine and cotinine on the vascular endothelial cell function. METHODS: Porcine common carotid arteries were cultured in a novel vascular perfusion system with nicotine or cotinine or as controls. After 24 h, vessels were precontracted with norepinephrine and subsequently relaxed with acetylcholine. Vessel diameters were recorded and analyzed. After culture, samples were taken for en face, immunohistochemistry, and RT-PCR for eNOS. Porcine coronary arteries were incubated as controls or with nicotine or cotinine and tested on a myograph system to measure contraction and relaxation. RESULTS: Porcine carotid arteries treated with nicotine and cotinine showed a 27.2% and a 41.2% reduction in endothelial-dependent relaxation, respectively, as compared to control vessels (P<0.05). Rings of coronary arteries treated with nicotine relaxed similarly to control rings while cotinine-treated rings failed to relax to endothelial-dependent stimulation. RT-PCR for eNOS mRNA showed a 23. 2 and a 24.1% reduction in eNOS expression for nicotine- and cotinine-treated vessels, respectively (P<0.01). Additionally, immunohistochemical staining for eNOS showed less dense staining on nicotine- and cotinine-treated vessels as compared to controls. En face preparations showed normal endothelial cell morphology in all groups, but cell density decreased slightly in vessels treated with nicotine and cotinine. CONCLUSION: These results indicate that cotinine may have even more effect on the impairment of endothelial-dependent vasorelaxation than nicotine for the regulation of vessel tone in porcine carotid and coronary arteries.