Introduction: We have previously shown that even one minute of exposure to secondhand smoke (SHS) of tobacco and marijuana can impair arterial flow-mediated dilation (FMD) in rats. The identity of specific constituents of SHS that cause vascular toxicity remains unclear. Nicotine is a vasoconstrictor that potentially can affect vascular function. Menthol is a cigarette additive popular with African Americans, women, youth, and other ethnicities. Menthol interacts with smoke constituents and irritants such as acrolein through receptor ion channels and also affects the metabolism rate of nicotine. Hypothesis: We tested three hypotheses pertaining to acute effects of brief exposures to SHS: (1) nicotine increases the extent of endothelial functional impairment; (2) menthol decreases the extent of endothelial functional impairment; and (3) there is an interaction between the effects of nicotine and menthol. Methods: We exposed 8 groups of rats (n=8-10/group) for 10 minutes to two different levels of SHS from the following four different types of research reference tobacco: conventional nicotine (~15 mg/g tobacco), reduced nicotine (~0.4 mg/g tobacco), and both conventional and reduced nicotine with added menthol (~1.15 mg/g tobacco) in a 2x2 factorial (ANOVA) design. We also examined a clean air negative control group (n=9). FMD was measured before and after exposures in each rat. To calculate FMD, the femoral artery diameter was measured with micro-ultrasound before and after a 5-min transient ischemia induced by an external vascular occluder. FMD was quantified as the % vasodilation. Results: FMD was not affected in the clean air controls (p=0.13). Higher nicotine levels were associated with increased % reduction of FMD impairment (19.22±9.53% vs. 41.07±4.54% (SD); p=0.047). Adding menthol was associated with decreased % reduction of FMD impairment (40.52±4.85% vs. 18.50±9.81%; p=0.048). These two effects were independent (p for nicotine x menthol interaction = 0.6). Conclusion: Reduction of nicotine level and addition of menthol each lessen the acute impairment of FMD in rats exposed briefly to tobacco SHS.