Cigarette mainstream smoke is a known risk factor for atherosclerosis. The treatment of rat aortic rings in vitro with aqueous solutions of mainstream cigarette smoke leads to endothelial dysfunction indicated by diminished vasorelaxation and enhanced vasoconstriction. The objective of this study was to evaluate whether nicotine, alone, has a similar effect. Rat aortic rings were incubated in vitro with nicotine to analyze the influence of nicotine on vessel function. Vasorelaxation and vasoconstriction properties were analyzed. Vasorelaxation was evoked with acetylcholine and vasoconstriction was evoked with norepinephrine. No significant effect of nicotine was observed. Experiments with cigarette smoke gas phase and particle phase containing different amounts of nicotine also indicated that nicotine does not affect the vasomotor function of rat aortic rings in vitro. The results of this study suggest that the endothelial dysfunction of rat aortic rings observed after treatment with aqueous solutions of cigarette mainstream smoke in vitro is not mediated by nicotine. Further research is necessary to identify the compounds that evoke this effect.